Antibodies to Olanzapine Haptens and Use Thereof

ABSTRACT

Disclosed is an antibody which binds to olanzapine, which can be used to detect olanzapine in a sample such as in a competitive immunoassay method. The antibody can be used in a lateral flow assay device for point-of-care detection of olanzapine, including multiplex detection of aripiprazole, olanzapine, quetiapine, and risperidone in a single lateral flow assay device.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/691,572, filed Aug. 21, 2012.

FIELD OF THE INVENTION

The present invention relates to the field of immunoassays, and inparticular to antibodies that bind to olanzapine which can be used inimmunoassays for detection of olanzapine.

BACKGROUND

Schizophrenia is a chronic and debilitating psychiatric disorderaffecting approximately 0.45-1% of the world's population (van Os, J.;Kapur, S. “Schizophrenia” Lancet 2009, 374, 635-645). The principalgoals of treatment are to achieve sustained remission from psychoticsymptoms, reduce the risk and consequences of relapse, and improvepatient functioning and overall quality of life. While many patientswith schizophrenia are able to achieve symptom stability with theavailable antipsychotic medications, poor adherence to medication is acommon reason for relapse with daily administered oral medications.Several studies (Abdel-Baki, A.; Ouellet-Plamondon, C.; Malla, A.“Pharmacotherapy Challenges in Patients with First-Episode Psychosis”Journal of Affective Disorders 2012, 138, S3-S14) investigating theoutcomes of non-compliance have shown that patients with schizophreniawho do not take their medication as prescribed have higher rates ofrelapse, hospital admission and suicide as well as increased mortality.It is estimated that 40 to 75% of patients with schizophrenia havedifficulty adhering to a daily oral treatment regimen (Lieberman, J. A.;Stroup, T. S.; McEvoy, J. P.; Swartz, M. S.; Rosenheck, R. A.; Perkins,D. O.; Keefe, R. S. E.; Davis, S. M.; Davis, C. E.; Lebowitz, B. D.;Severe, J.; Hsiao, J. K. “Effectiveness of Antipyschotic Drugs inPatients with Chronic Schizophrenia” New England Journal of Medicine2005, 353(12), 1209-1223).

Therapeutic drug monitoring (TDM) is the quantification of serum orplasma concentrations of drugs, including anti-psychotic drugs, fortreatment monitoring and optimization. Such monitoring permits, forexample, the identification of patients that are not adhering to theirmedication regimen, that are not achieving therapeutic doses, that arenon-responsive at therapeutic doses, that have suboptimal tolerability,that have pharmacokinetic drug-drug interactions, or that have abnormalmetabolism resulting in inappropriate plasma concentrations.Considerable individual variability exists in the patient's ability toabsorb, distribute, metabolize, and excrete anti-psychotic drugs. Suchdifferences can be caused by concurrent disease, age, concomitantmedication or genetic peculiarities. Different drug formulations canalso influence the metabolism of anti-psychotic drugs. TDM permits doseoptimization for individual patients, improving therapeutic andfunctional outcomes. TDM further permits a prescribing clinician toensure compliance with prescribed dosages and achievement of effectiveserum concentrations.

To date, methods for determining the levels of serum or plasmaconcentrations of anti-psychotic drugs involve the use of liquidchromatography (LC) with UV or mass spectrometry detection, andradioimmunoassays (see, for example, Woestenborghs et al., 1990 “On theselectivity of some recently developed RIA's” in Methodological Surveysin Biochemistry and Analysis 20:241-246. Analysis of Drugs andMetabolites, Including Anti-infective Agents; Heykants et al., 1994 “ThePharmacokinetics of Risperidone in Humans: A Summary”, J Clin Psychiatry55/5, suppl:13-17; Huang et al., 1993 “Pharmacokinetics of the novelanti-psychotic agent risperidone and the prolactin response in healthysubjects”, Clin Pharmacol Ther 54:257-268). Radioimmunoassays detect oneor both of risperidone and paliperidone. Salamone et al. in U.S. Pat.No. 8,088,594 disclose a competitive immunoassay for risperidone usingantibodies that detect both risperidone and paliperidone but notpharmacologically inactive metabolites. The antibodies used in thecompetitive immunoassay are developed against a particular immunogen. IDLabs Inc. (London, Ontario, Canada) markets an ELISA for olanzapine,another anti-psychotic drug, which also utilizes a competitive format.The Instructions For Use indicate that the assay is designed forscreening purposes and intended for forensic or research use, and isspecifically not intended for therapeutic use. The Instructionsrecommend that all positive samples should be confirmed with gaschromatography/mass spectrometry (GC-MS), and indicate that the antibodyused detects olanzapine and clozapine (see ID Labs Inc., “InstructionsFor Use Data Sheet IDEL-F083”, Rev. Date Aug. 8, 2011). Some of thesemethods, namely HPLC and GC/MS, can be expensive and labor-intensive,and are generally only performed in large or specialty labs having theappropriate equipment.

A need exists for other methods for determining the levels ofanti-psychotic drugs, particularly methods that can be performed in aprescribing clinician's office (where the treatment for an individualpatient can be adjusted accordingly in a much more timely manner) and inother medical settings lacking LC or GC/MS equipment or requiring rapidtest results.

Olanzapine is:

SUMMARY OF THE INVENTION

The present invention is directed to an isolated antibody or a bindingfragment thereof, which binds to olanzapine and which: (i) is generatedin response to a conjugate of a compound of Formula I and an immunogeniccarrier; or (ii) competes for an epitope which is the same as an epitopebound by the antibody of (i).

wherein:

R¹ is H,

CH₂NH₂, CH₂NHC(O)(CH₂)_(m)CO₂H, or Z—(Y)_(p)-G;

R² is H,

CH₂NH₂, CH₂NHC(O)(CH₂)_(m)CO₂H, or Z—(Y)_(p)-G;R³ is H, or W—(Y)_(p)-G; provided that two of R¹, R², R³ must be H, andfurther provided that R¹, R² and R³ may not all be H simultaneously;wherein:Z is selected from the group consisting of:—N(R⁴)—, —O—, —S—, -alkyl-, -alkoxyalkyl-, -aminoalkyl-, -thioalkyl-,-heteroalkyl-, alkylcarbonyl-,

wherein:W is selected from the group consisting of:—C(O)—, -alkyl-, -alkoxyalkyl-, -aminoalkyl-, -thioalkyl-,-heteroalkyl-, -alkylcarbonyl-,

—N(R⁴)—,

R⁴ is H, an alkyl group, cycloalkyl group, araalkyl group or substitutedor unsubstituted aryl group;Y is an organic spacer group;G is a functional linking group capable of binding to a carrier;p is 0, or 1;m is 1, 2, 3, 4, or 5;n is 1, 2, 3, 4, or 5.

Presently preferred embodiments of the antibody of the subject inventionare the antibodies designated 35 and 61 generated against the compoundhaving Formula II and the antibodies designated 3F11 and 4G9-1 generatedagainst the compound having Formula III. Another suitable immunogen isthe compound having Formula IV.

The antibodies of the subject invention can be provided in assay kitsand assay devices, with a presently preferred device being a lateralflow assay device which provides for point-of-care analysis.

The invention further provides a method of producing an antibody whichbinds to olanzapine, the method comprising: (i) selecting a host cellfor antibody production; and (ii) inoculating the host with a conjugateof a compound of Formula I and an immunogenic carrier, wherein the hostproduces an antibody which binds to olanzapine. Further provided is amethod of producing a hybridoma cell line capable of producing amonoclonal antibody which binds to olanzapine. The method comprises: (i)selecting a host for antibody production; (ii) inoculating the host witha conjugate of a compound of Formula I and an immunogenic carrier; (iii)fusing a cell line from the inoculated host with a continuously dividingcell to create a fused cell capable of producing a monoclonal antibodywhich binds to olanzapine; and (iv) cloning the fused cell so as toobtain a hybridoma cell line.

The invention further provides a method of detecting olanzapine in asample. The method comprises: (i) contacting a sample with an antibodyaccording to the subject invention which is labeled with a detectablemarker, wherein the labeled antibody and olanzapine present in thesample form a labeled complex; and (ii) detecting the labeled complex soas to detect olanzapine in the sample.

Further provided is a competitive immunoassay method for detectingolanzapine in a sample. The method comprises: (i) contacting a samplewith an antibody according to the subject invention, and with olanzapineor a competitive binding partner of olanzapine, wherein one of theantibody and the olanzapine or competitive binding partner thereof islabeled with a detectable marker, and wherein sample olanzapine competeswith the olanzapine or competitive binding partner thereof for bindingto the antibody; and (ii) detecting the label so as to detect sampleolanzapine.

Further objects, features and advantages of the present invention willbe apparent to those skilled in the art from detailed consideration ofthe preferred embodiments that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-3 show Competitive ELISA results generated with three differentmouse fusion 11.1 hybridomas;

FIG. 4 shows the competitive immunoassay format used on a lateral flowassay device;

FIG. 5 shows a typical dose response curve generated with olanzapineantibody clone 35;

FIG. 6 shows a typical dose response curve generated with olanzapineantibody clone 61;

FIG. 7 shows a typical dose response curve generated with olanzapineantibody 3F11;

FIG. 8 shows the chip design of a lateral flow assay device according tothe subject invention;

FIG. 9 shows a typical dose response curve for an aripiprazole positivecontrol generated with antibody 5C7 and a labeled aripiprazolecompetitive binding partner;

FIG. 10 shows a typical dose response curve for an olanzapine positivecontrol generated with antibody 4G9-1 and a labeled olanzapinecompetitive binding partner;

FIG. 11 shows a typical dose response curve for a quetiapine positivecontrol generated with antibody 11 and a labeled quetiapine competitivebinding partner;

FIG. 12 shows a typical dose response curve for a risperidone positivecontrol generated with antibody 5-9 and a labeled risperidonecompetitive binding partner;

FIG. 13 shows a typical dose response curve for a sample containingaripiprazole generated with aripiprazole antibody 5C7 in the presence oflabeled aripiprazole competitive binding partner, with no dose responsecurve for olanzapine, quetiapine, or risperidone in the presence of alabeled competitive binding partner for each;

FIG. 14 shows a typical dose response curve for a sample containingolanzapine generated with olanzapine antibody 4G9-1 in the presence of alabeled olanzapine competitive binding partner, with no dose responsecurve for aripiprazole, quetiapine, or risperidone in the presence of alabeled competitive binding partner for each;

FIG. 15 shows a typical dose response curve for a sample containingquetiapine generated with quetiapine antibody 11 in the presence of alabeled quetiapine competitive binding partner, with no dose responsecurve for aripiprazole, olanzapine, or risperidone in the presence of alabeled competitive binding partner for each;

FIG. 16 shows a typical dose response curve for a sample containingrisperidone generated with risperidone antibody 5-9 in the presence of alabeled risperidone competitive binding partner, with no dose responsecurve for aripiprazole, olanzapine, or quetiapine in the presence of alabeled competitive binding partner for each;

FIG. 17 shows a typical dose response curve for a sample containingaripiprazole generated with aripiprazole antibody 5C7 in the presence ofa labeled aripiprazole competitive binding partner, with no doseresponse curve for olanzapine, quetiapine, or risperidone in thepresence of antibody and labeled competitive binding partner for each;

FIG. 18 shows a typical dose response curve for a sample containingolanzapine generated with olanzapine antibody 4G9-1 in the presence of alabeled olanzapine competitive binding partner, with no dose responsecurve for aripiprazole, quetiapine, or risperidone in the presence ofantibody and labeled competitive binding partner for each;

FIG. 19 shows a typical dose response curve for a sample containingquetiapine generated with quetiapine antibody 11 in the presence oflabeled quetiapine competitive binding partner, with no dose responsecurve for aripiprazole, olanzapine, or risperidone in the presence ofantibody and labeled competitive binding partner for each;

FIG. 20 shows a typical dose response curve for a sample containingrisperidone generated with risperidone antibody 5-9 in the presence of alabeled risperidone competitive binding partner, with no dose responsecurve for aripiprazole, olanzapine, or quetiapine in the presence ofantibody and labeled competitive binding partner for each;

FIG. 21 shows a comparison of the aripiprazole dose response curvegenerated as a positive control to the aripiprazole dose response curvegenerated in the multiplex format;

FIG. 22 shows a comparison of the olanzapine dose response curvegenerated as a positive control to the olanzapine dose response curvegenerated in the multiplex format;

FIG. 23 shows a comparison of the quetiapine dose response curvegenerated as a positive control to the quetiapine dose response curvegenerated in the multiplex format; and

FIG. 24 shows a comparison of the risperidone dose response curvegenerated as a positive control to the risperidone dose response curvegenerated in the multiplex format.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The invention provides an isolated antibody which binds to olanzapine.The invention further provides an assay kit and an assay devicecomprising the antibody. Also provided are methods of producing theantibody and of producing a hybridoma cell line capable of producing theantibody. Further provided is a method of detecting olanzapine in asample, including a competitive immunoassay method.

In one embodiment, the present invention is directed to an isolatedantibody or a binding fragment thereof, which binds to olanzapine andwhich: (i) is generated in response to a conjugate of a compound ofFormula I and an immunogenic carrier; or (ii) competes for an epitopewhich is the same as an epitope bound by the antibody of (i).

Formula I:

wherein:

R¹ is H,

CH₂NH₂, CH₂NHC(O)(CH₂)_(m)CO₂H, or Z—(Y)_(p)-G;

R² is H,

CH₂NH₂, CH₂NHC(O)(CH₂)_(m)CO₂H, or Z—(Y)_(p)-G;R³ is H, or W—(Y)_(p)-G; provided that two of R¹, R², R³ must be H, andfurther provided that R¹, R² and R³ may not all be H simultaneously;wherein:Z is selected from the group consisting of:—N(R⁴)—, —O—, —S—, -alkyl-, -alkoxyalkyl-, -aminoalkyl-, -thioalkyl-,-heteroalkyl-, alkylcarbonyl-,

wherein:W is selected from the group consisting of:—C(O)—, -alkyl-, -alkoxyalkyl-, -aminoalkyl-, -thioalkyl-,-heteroalkyl-, -alkylcarbonyl-, —N(R⁴)—,

R⁴ is H, an alkyl group, cycloalkyl group, araalkyl group or substitutedor unsubstituted aryl group;Y is an organic spacer group;G is a functional linking group capable of binding to a carrier;p is 0, or 1;m is 1, 2, 3, 4, or 5;n is 1, 2, 3, 4, or 5.

In a further embodiment, the present invention is directed to anisolated antibody or a binding fragment thereof, which binds toolanzapine and which: (i) is generated in response to a conjugate of acompound of Formula I and an immunogenic carrier; or (ii) competes foran epitope which is the same as an epitope bound by the antibody of (i);wherein:

R¹ is H,

CH₂NH₂, CH₂NHC(O)(CH₂)_(m)CO₂H, or Z—(Y)_(p)-G;

R² is H,

CH₂NH₂, CH₂NHC(O)(CH₂)_(m)CO₂H, or Z—(Y)_(p)-G; provided that either R¹or R² must be H, and further provided that both R¹ and R² may not be Hsimultaneously;

R³ is H;

wherein:Z is selected from the group consisting of:—N(R⁴)—, —O—, —S—, -alkyl-, -alkoxyalkyl-, -aminoalkyl-, -thioalkyl-,-heteroalkyl-, -alkylcarbonyl-,

R⁴ is H, an alkyl group, cycloalkyl group, araalkyl group or substitutedor unsubstituted aryl group;Y is an organic spacer group;G is a functional linking group capable of binding to a carrier;p is 0, or 1;m is 1, 2, 3, 4, or 5;n is 1, 2, 3, 4, or 5.

In a further embodiment, the present invention is directed to anisolated antibody or a binding fragment thereof, which binds toolanzapine and which: (i) is generated in response to a conjugate of acompound of Formula I and an immunogenic carrier; or (ii) competes foran epitope which is the same as an epitope bound by the antibody of (i);wherein:

R¹ is H, or CH₂NH—(Y)_(p)-G;

R² is H, or CH₂NH—(Y)_(p)-G; provided that either R¹ or R² must be H,and further provided that both R¹ and R² may not be H simultaneously;

R³ is H,

wherein:Y is an organic spacer group;G is a functional linking group capable of binding to a carrier;p is 1.

In a further embodiment, the present invention is directed to anisolated antibody or a binding fragment thereof, which binds toolanzapine and which: (i) is generated in response to a conjugate of acompound of Formula I and an immunogenic carrier; or (ii) competes foran epitope which is the same as an epitope bound by the antibody of (i);wherein:

R¹ is H,

CH₂NH₂, or CH₂NHC(O)(CH₂)_(m)CO₂H;

R² is H,

CH₂NH₂, or CH₂NHC(O)(CH₂)_(m)CO₂H; provided that either R¹ or R² must beH, and further provided that both R¹ and R² may not be H simultaneously;

R³ is H;

m is 1, 2, 3, 4, or 5;n is 1, 2, 3, 4, or 5.

In a further embodiment, the present invention is directed to anisolated antibody or a binding fragment thereof, which binds toolanzapine and which: (i) is generated in response to a conjugate of acompound of Formula I and an immunogenic carrier; or (ii) competes foran epitope which is the same as an epitope bound by the antibody of (i);wherein:

R¹ is H,

CH₂NH₂, or CH₂NHC(O)(CH₂)_(m)CO₂H;

R² is H,

CH₂NH₂, or CH₂NHC(O)(CH₂)_(m)CO₂H; provided that either R¹ or R² must beH, and further provided that both R¹ and R² may not be H simultaneously;

R³ is H;

m is 1, 2, 3, 4, or 5;n is 1, 2, 3, 4, or 5.

In a preferred embodiment, the present invention is directed to anisolated antibody or a binding fragment thereof, which binds toolanzapine and which: (i) is generated in response to a conjugate of acompound of Formula V and an immunogenic carrier; or (ii) competes foran epitope which is the same as an epitope bound by the antibody of (i).

In a preferred embodiment, the present invention is directed to anisolated antibody or a binding fragment thereof, which binds toolanzapine and which: (i) is generated in response to a conjugate of acompound of Formula VI and an immunogenic carrier; or (ii) competes foran epitope which is the same as an epitope bound by the antibody of (i).

In a preferred embodiment, the present invention is directed to anisolated antibody or a binding fragment thereof, which binds toolanzapine and which: (i) is generated in response to a conjugate of acompound of Formula VII and an immunogenic carrier; or (ii) competes foran epitope which is the same as an epitope bound by the antibody of (i).

In a preferred embodiment, the present invention is directed to anisolated antibody or a binding fragment thereof, which binds toolanzapine and which: (i) is generated in response to a conjugate of acompound of Formula VIII and an immunogenic carrier; or (ii) competesfor an epitope which is the same as an epitope bound by the antibody of(i).

In a preferred embodiment, the present invention is directed to anisolated antibody or a binding fragment thereof, which binds toolanzapine and which: (i) is generated in response to a conjugate of acompound of Formula IX and an immunogenic carrier; or (ii) competes foran epitope which is the same as an epitope bound by the antibody of (i).

In a preferred embodiment, the present invention is directed to anisolated antibody or a binding fragment thereof, which binds toolanzapine and which: (i) is generated in response to a conjugate of acompound of Formula X and an immunogenic carrier; or (ii) competes foran epitope which is the same as an epitope bound by the antibody of (i).

Preferably, the antibody of the subject invention is generated inresponse to a conjugate of a compound selected from the compounds of:Formula I, Formula V, Formula VI, Formula VII, Formula VIII, Formula IX,and Formula X; and an immunogenic carrier.

Further details of the compounds described by the formulas above and theconjugates formed by the compounds and an immunogenic carrier areprovided in the section below entitled “Compounds, Conjugates andImmunogens”.

Further details of the antibodies of the subject invention are providedin the section below entitled “Antibodies”.

The subject invention further provides an assay kit comprising theantibody, as well as an assay device comprising the antibody.Preferably, the assay device is a lateral flow assay device. Furtherdetails of the assay kits and assay devices are provided below in thesection entitled “Assay Kits and Devices”.

The invention further provides a method of producing an antibody whichbinds to olanzapine, the method comprising: (i) selecting a host cellfor antibody production; and (ii) inoculating the host with a conjugateof a compound of Formula I and an immunogenic carrier, wherein the hostproduces an antibody which binds to olanzapine. In additionalembodiments, the conjugate used in the method can be a conjugate of acompound selected from the compounds of: Formula V, Formula VI, FormulaVII, Formula VIII, Formula IX, and Formula X; and an immunogeniccarrier. Further details on the production of the antibodies of thesubject invention are provided in the section below entitled“Antibodies”.

Further provided is a method of producing a hybridoma cell line capableof producing a monoclonal antibody which binds to olanzapine. The methodcomprises: (i) selecting a host for antibody production; (ii)inoculating the host with a conjugate of a compound of Formula I and animmunogenic carrier; (iii) fusing a cell line from the inoculated hostwith a continuously dividing cell to create a fused cell capable ofproducing a monoclonal antibody which binds to olanzapine; and (iv)cloning the fused cell so as to obtain a hybridoma cell line. Inadditional embodiments, the conjugate used in the method can be aconjugate of a compound selected from the compounds of: Formula V,Formula VI, Formula VII, Formula VIII, Formula IX, and Formula X; and animmunogenic carrier. Further details of the production of hybridomas inaccordance with the subject invention are provided in the section belowentitled “Antibodies”.

The invention further provides a method of detecting olanzapine in asample. The method comprises: (i) contacting a sample with an antibodyaccording to the subject invention which is labeled with a detectablemarker, wherein the labeled antibody and olanzapine present in thesample form a labeled complex; and (ii) detecting the labeled complex soas to detect olanzapine in the sample. Further details of the method ofdetecting olanzapine in accordance with the subject invention areprovided in the section below entitled “Immunoassays”.

Further provided is a competitive immunoassay method for detectingolanzapine in a sample. The method comprises: (i) contacting a samplewith an antibody according to the subject invention, and with olanzapineor a competitive binding partner of olanzapine, wherein one of theantibody and the olanzapine or competitive binding partner thereof islabeled with a detectable marker, and wherein sample olanzapine competeswith the olanzapine or competitive binding partner thereof for bindingto the antibody; and (ii) detecting the label so as to detect sampleolanzapine. Further details of the competitive immunoassay method ofdetecting olanzapine in accordance with the subject invention areprovided in the section below entitled “Immunoassays”.

In a preferred embodiment of the subject invention, the detection ofolanzapine is accompanied by the detection of one or more analytes inaddition to olanzapine. Preferably the one or more analytes areanti-psychotic drugs other than olanzapine, and more preferably theanti-psychotic drugs other than olanzapine are selected from the groupconsisting of: aripiprazole, risperidone, paliperidone, quetiapine, andmetabolites thereof.

As discussed above, the antibodies of the subject invention can be usedin assays to detect the presence and/or amount of the anti-psychoticdrug in patient samples. Such detection permits therapeutic drugmonitoring enabling all of the benefits thereof. Detection of levels ofanti-psychotic drugs may be useful for many purposes, each of whichrepresents another embodiment of the subject invention, including:determination of patient adherence or compliance with prescribedtherapy; use as a decision tool to determine whether a patient should beconverted from an oral anti-psychotic regimen to a long-actinginjectable anti-psychotic regimen; use as a decision tool to determineif the dose level or dosing interval of oral or injectableanti-psychotics should be increased or decreased to ensure attainment ormaintenance of efficacious or safe drug levels; use as an aid in theinitiation of anti-psychotic drug therapy by providing evidence of theattainment of minimum pK levels; use to determine bioequivalence ofanti-psychotic drug in multiple formulations or from multiple sources;use to assess the impact of polypharmacy and potential drug-druginteractions; and use as an indication that a patient should be excludedfrom or included in a clinical trial and as an aid in the subsequentmonitoring of adherence to clinical trial medication requirements.

Compounds, Conjugates and Immunogens

In relation to the compounds and conjugates and immunogens, thefollowing abbreviations are used: AMAS is N-(α-maleimidoacetoxy)succinimide ester; BTG is bovine thyroglobulin; Bu₃N is tributylamine;DCC is dicyclohexylcarbodiimide; DCM is dichloromethane; DIEA isdiisopropylethylamine; DMF is N,N-dimethylformamide; DMSO isdimethylsulfoxide; EDTA is ethylenediaminetetraceticacid; KLH is keyholelimpet hemocyanin; SATA is N-succinimidyl S-acetylthioacetate; TEA istriethylamine; THF is tetrahydrofuran; TFA is trifluoroacetic acid; r.t.is room temperature; DIC is diisopropylcarbodiimide; DMAP isN,N-dimethyl-4-aminopyridine; EDC is1-ethyl-3(3-dimethylaminopropyl)carbodiimidehydrochloride; NHS isN-hydroxysuccinimide; TFP is Tetrafluorophenyl; PNP is p-nitrophenyl;TBTU is O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborate; HOBT is N-Hydroxybenzotriazole; DEPBT is3-(diethoxyphosphoryloxy)-1,2,3-benzotrazin-4(3H)-one; BOP—CI isBis(2-oxo-3-oxazolidinyl)phosphonic chloride; and DTT isdithioerythritol.

The term “conjugate” refers to any substance formed from the joiningtogether of separate parts. Representative conjugates include thoseformed by the joining together of a small molecule, such as thecompounds of Formula I, and a large molecule, such as a carrier or apolyamine polymer, particularly a protein. In the conjugate the smallmolecule may be joined at one or more active sites on the largemolecule.

The term “hapten” refers to a partial or incomplete antigen. A hapten isa protein-free substance, which is not capable of stimulating antibodyformation, but which does react with antibodies. The antibodies areformed by coupling a hapten to a high molecular weight immunogeniccarrier, and then injecting this coupled product, i.e., an immunogen,into a human or animal subject.

The term “immunogen” refers to a substance capable of eliciting,producing, or generating an immune response in an organism.

An “immunogenic carrier,” as used herein, is an immunogenic substance,commonly a protein, that can join at one or more positions with haptens,thereby enabling the production of antibodies that can bind with thesehaptens. Examples of immunogenic carrier substances include, but are notlimited to, proteins, glycoproteins, complex polyamino-polysaccharides,particles, and nucleic acids that are recognized as foreign and therebyelicit an immunologic response from the host. Thepolyamino-polysaccharides may be prepared from polysaccharides using anyof the conventional means known for this preparation.

Various protein types may be employed as immunogenic carriers, includingwithout limitation, albumins, serum proteins, lipoproteins, etc.Illustrative proteins include bovine serum albumin, keyhole limpethemocyanin, egg ovalbumin, bovine thyroglobulin, fraction V human serumalbumin, rabbit albumin, pumpkin seed globulin, diphtheria toxoid,tetanus toxoid, botilinus toxin, succinylated proteins, and syntheticpoly(aminoacids) such as polylysine.

Immunogenic carriers can also include poly amino-polysaccharides, whichare a high molecular weight polymers built up by repeated condensationsof monosaccharides. Examples of polysaccharides are starches, glycogen,cellulose, carbohydrate gums such as gum arabic, agar, and so forth. Thepolysaccharide also contains poly(amino acid) residues and/or lipidresidues.

The immunogenic carrier can also be a poly(nucleic acid) either alone orconjugated to one of the above mentioned poly(amino acids) orpolysaccharides.

The immunogenic carrier can also include solid particles. The particlesare generally at least about 0.02 microns (μm) and not more than about100 μm, and usually about 0.05 μm to 10 μm in diameter. The particle canbe organic or inorganic, swellable or non-swellable, porous ornon-porous, optimally of a density approximating water, generally fromabout 0.7 to 1.5 g/mL, and composed of material that can be transparent,partially transparent, or opaque. The particles can be biologicalmaterials such as cells and microorganisms, including non-limitingexamples such as erythrocytes, leukocytes, lymphocytes, hybridomas,Streptococcus, Staphylococcus aureus, E. coli, and viruses. Theparticles can also be comprised of organic and inorganic polymers,liposomes, latex, phospholipid vesicles, or lipoproteins.

The term “derivative” refers to a chemical compound or molecule madefrom a parent compound by one or more chemical reactions.

The term “analogue” of a chemical compound refers to a chemical compoundthat contains a chain of carbon atoms and the same particular functionalgroups as a reference compound, but the carbon chain of the analogue islonger or shorter than that of the reference compound.

A “label,” “detector molecule,” “reporter” or “detectable marker” is anymolecule which produces, or can be induced to produce, a detectablesignal. The label can be conjugated to an analyte, immunogen, antibody,or to another molecule such as a receptor or a molecule that can bind toa receptor such as a ligand, particularly a hapten or antibody. A labelcan be attached directly or indirectly by means of a linking or bridgingmoiety. Non-limiting examples of labels include radioactive isotopes(e.g., ¹²⁵I), enzymes (e.g. β-galactosidase, peroxidase), enzymefragments, enzyme substrates, enzyme inhibitors, coenzymes, catalysts,fluorophores (e.g., rhodamine, fluorescein isothiocyanate or FITC, orDylight 649), dyes, chemiluminescers and luminescers (e.g., dioxetanes,luciferin), or sensitizers.

As used herein, a “spacer” refers to a portion of a chemical structurewhich connects two or more substructures such as haptens, carriers,immunogens, labels or binding partners through a functional linkinggroup. These spacer groups are composed of the atoms typically presentand assembled in ways typically found in organic compounds and so may bereferred to as “organic spacing groups”. The chemical building blocksused to assemble the spacers will be described hereinafter in thisapplication. Among the preferred spacers are straight or branched,saturated or unsaturated carbon chains. These carbon chains may alsoinclude one or more heteroatoms within the chain, one or moreheteroatoms replacing one or more hydrogens of any carbon atom in thechain, or at the termini of the chains. By “heteroatoms” is meant atomsother than carbon which are chosen from the group consisting of oxygen,nitrogen, phosphorous and sulfur, wherein the nitrogen, phosphorous andsulfur atoms may exist in any oxidation state and may have carbon orother heteroatoms bonded to them. The spacer may also include cyclic oraromatic groups as part of the chain or as a substitution on one of theatoms in the chain.

The number of atoms in the spacing group is determined by counting theatoms other than hydrogen. The number of atoms in a chain within aspacing group is determined by counting the number of atoms other thanhydrogen along the shortest route between the substructures beingconnected. Preferred chain lengths are between 1 to 20 atoms.

A “functional linking group” refers to a reactive group that is presenton a hapten and may be used to provide an available reactive sitethrough which the hapten portion may be coupled to another moietythrough formation of a covalent chemical bond to produce a conjugate ofa hapten with another moiety (such as a label or carrier). The haptenmay be linked in this way to a moiety such as biotin to form acompetitive binding partner.

Spacer groups may be used to link the hapten to the carrier. Spacers ofdifferent lengths allow one to attach the hapten with differingdistances from the carrier for presentation to the immune system of theanimal or human being immunized for optimization of the antibodyformation process. Attachment to different positions in the haptenmolecule allows the opportunity to present specific sites on the haptento the immune system to influence antibody recognition. The spacer maycontain hydrophilic solubilizing groups to make the hapten derivativemore soluble in aqueous media. Examples of hydrophilic solubilizinggroups include but are not limited to polyoxyalkyloxy groups, forexample, polyethylene glycol chains; hydroxyl, carboxylate and sulfonategroups.

The term “nucleophilic group” or “nucleophile” refers to a species thatdonates an electron-pair to form a chemical bond in a reaction. The term“electrophilic group” or “electrophile” refers to a species that acceptsan electron-pair from a nucleophile to form a chemical bond in areaction.

The term “substituted” refers to substitution of an atom or group ofatoms in place of a hydrogen atom on a carbon atom in any position onthe parent molecule. Non limiting examples of substituents includehalogen atoms, amino, hydroxy, carboxy, alkyl, aryl, heteroalkyl,heteroaryl, cyano, alkoxy, nitro, aldehyde and ketone groups.

The term “alkyl” refers to saturated or unsaturated linear and branchedchain radicals of up to 12 carbon atoms, unless otherwise indicated, andis specifically intended to include radicals having any degree or levelof saturation. Alkyl includes, but is not limited to, methyl, ethyl,propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl,isopentyl, hexyl, isohexyl, heptyl, octyl, 2,2,4-trimethylpentyl, nonyl,decyl, undecyl and dodecyl.

The term “cycloalkyl” refers to a saturated or partially unsaturatedmonocyclic or bicyclic hydrocarbon ring radical composed of from 3 to 10carbon atoms. Alkyl substituents may optionally be present on the ring.Examples include cyclopropyl, 1,1-dimethyl cyclobutyl,1,2,3-trimethylcyclopentyl, cyclohexyl and cyclohexenyl.

The term “heteroalkyl” refers to an alkyl group that includes one ormore heteroatoms within the chain, one or more heteroatoms replacing oneor more hydrogens of any carbon atom in the chain, or at termini of thechains.

The term “aminoalkyl” refers to at least one primary or secondary aminogroup bonded to any carbon atom along an alkyl chain.

The term “alkoxy” refers to straight or branched chain radicals of up to12 carbon atoms, unless otherwise indicated, bonded to an oxygen atom.Examples include but are not limited to methoxy, ethoxy, propoxy,isopropoxy and butoxy.

The term “alkoxyalkyl” refers to at least one alkoxy group bonded to anycarbon atom along an alkyl chain.

The term “thioalkyl” refers to at least one sulfur group bonded to anycarbon atom along an alkyl chain. The sulfur group may be at anyoxidation state and includes sulfoxides, sulfones and sulfates.

The term “carboxylate group” includes carboxylic acids and alkyl,cycloalkyl, aryl or aralkyl carboxylate esters.

The term “alkylcarbonyl” refers to a group that has a carbonyl groupbonded to any carbon atom along an alkyl chain.

The term “heteroaryl” refers to 5- to 7-membered mono- or 8- to10-membered bicyclic aromatic ring radicals, any ring of which mayconsist of from one to four heteroatoms selected from N, O or S wherethe nitrogen and sulfur atoms can exist in any allowed oxidation state.Examples include benzimidazolyl, benzothiazolyl, benzothienyl,benzoxazolyl, furyl, imidazolyl, isothiazolyl, isoxazolyl, oxazolyl,pyrazinyl, pyrazolyl, pyridyl, pyrimidinyl, pyrrolyl, quinolinyl,thiazolyl and thienyl.

The term “aryl” refers to monocyclic or bicyclic aromatic ring radicalscontaining from 6 to 12 carbons in the ring. Alkyl substituents mayoptionally be present on the ring. Examples include phenyl, biphenyl andnapththalene.

The term “aralkyl” refers to a C₁₋₆ alkyl group containing an arylsubstituent. Examples include benzyl, phenylethyl or 2-naphthylmethyl.

The term “acyl” refers to the group —C(O)R_(a), where R_(a) is hydrogen,alkyl, cycloalkyl, heteroalkyl, aryl, aralkyl and heteroaryl. An“acylating agent” adds the —C(O)R_(a) group to a molecule.

The term “sulfonyl” refers to the group —S(O)₂R_(b), where R_(b) ishydrogen, alkyl, cycloalkyl, heteroalkyl, haloalkyl, aryl, aralkyl andheteroaryl. A “sulfonylating agent” adds the —S(O)₂R_(a) group to amolecule.

Spacers bearing reactive functional linking groups for the attachment ofhaptens to carrier moieties may be prepared by a wide variety ofmethods. The spacer may be formed using a molecule that isdifferentially functionalized or activated with groups at either end toallow selective sequential reaction with the hapten and the carrier, butthe same reactive moiety may also be used at both ends. The groupsselected for reaction with the hapten and the functional linking groupto be bound to the carrier are determined by the type of functionalityon the hapten and the carrier that the hapten is to be bonded with.Spacers and methods of attachment to haptens and carriers include butare not limited to those described by Brinkley, M., A., BioconjugateChem. 1992, 3:2-13, Hermanson, Greg T., Bioconjugate Techniques,Academic Press, London, Amsterdam, Burlington, Mass., USA, 2008 andThermo Scientific Pierce Crosslinking Technical Handbook; available fordownload or hard copy request from Thermo Scientific 3747 N Meridian Rd,Rockford, Ill. USA 61101, ph 800-874-3723 or at:http://www.piercenet.com/ and references within. Many differentiallyactivated molecules for formation of spacer groups are commerciallyavailable from vendors, for example Thermo Scientific.

For haptens bearing an amino group, modes of attachment of the spacer tothe hapten include reaction of the amine on the hapten with a spacerbuilding block bearing an acyl halide or active ester. “Active esters”are defined as esters that undergo reaction with a nucleophilic group,for example an amino group, under mild conditions to form a stablelinkage. A stable linkage is defined as one that remains intact underconditions of further use, for example subsequent synthetic steps, useas an immunogen, or in a biochemical assay. A preferred example of astable linkage is an amide bond. Active esters and methods of formationare described by Benoiton, N. L., in Houben-Weyl, Methods of OrganicChemistry, Thieme Stuttgart, N.Y., vol E22 section 3.2:443 and Benoiton,N. L., Chemistry of Peptide Synthesis, Taylor and Francis, N.Y., 2006.Preferred active esters include p-nitrophenyl ester (PNP),N-hydroxysuccinimide ester (NHS) and tetrafluorophenyl ester (TFP). Acylhalides may be prepared by many methods known to one skilled in the artfor example, reaction of the carboxylic acid with thionyl chloride oroxalyl chloride, see: Fieser, L. F. and Fieser, M. Reagents for OrganicSynthesis, John Wiley and Sons, NY, 1967 and references within. Thesemay be converted to other active esters such as p-nitrophenyl esters(PNP) which may also be used in active bi-functional spacers asdescribed by Wu et. al, Organic Letters, 2004, 6 (24):4407.N-hydroxysuccinimide (NHS) esters may be prepared by reaction ofN,N-disuccinimidyl carbonate (CAS 74124-79-1) with the carboxylic acidof a compound in the presence of an organic base such as triethylamineor diisopropylethylamine in an aprotic solvent under anhydrousconditions as described in Example 35 of WO2012012595 or by usingN-hydroxysuccinimide and dicyclohexylcarbodiimide (DCC) or otherdehydrating agent, under anhydrous conditions. Tetrafluorophenyl esters(TFP) may be prepared by reaction of carboxylic acids with2,3,5,6-tetrafluorophenyltrifluoroacetate in the presence of an organicbase such as triethylamine or diisopropylethylamine in an aproticsolvent under anhydrous conditions as reported by Wilbur, et. al,Bioconjugate Chem., 2004, 15(1):203. One skilled in the art willrecognize that spacers shown in Table 1, among others, can be obtainedusing known methods and attached to amino-bearing haptens utilizingroutine optimization of reaction conditions. These spacers allowattachment of the hapten to a thiol group on a carrier.

TABLE 1

Reasonable values for m and n are between 1 and 10

Direct coupling of the amine on the hapten and a carboxylic acidfunctionality on the spacer building block in the presence of a couplingagent may also be used as a mode of attachment. Preferred reagents arethose typically used in peptide synthesis. Peptide coupling reagentsinclude but are not limited toO-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate(TBTU, CAS #125700-67-6), see: Pruhs, S., Org. Process. Res. Dev. 2006,10:441; N-Hydroxybenzotriazole (HOBT, CAS #2592-95-2) with acarbodiimide dehydrating agent, for example N—N-dicyclohexylcarbodiimide(DCC), diisopropylcarbodiimide (DIC), or1-ethyl-3(3-dimethylaminopropyl)carbodiimidehydrochloride (EDC), see:König W., Geiger, R. Chem. Ber., 1970, 103 (3):788;3-(diethoxyphosphoryloxy)-1,2,3-benzotrazin-4(3H)-one (DEPBT,CAS#165534-43-0), see: Liu, H. et. al., Chinese Chemical Letters, 2002,13(7):601; Bis(2-oxo-3-oxazolidinyl)phosphonic chloride; (BOP—CI,CAS#68641-49-6), see: Diago-Meseguer, J et. al. Synthesis, 1980,7:547-51 and others described in detail by Benoiton in Chemistry ofPeptide Synthesis, CRC Press, Boca Raton, Fla., 2005, Chapter 2, and thetechnical bulletin provided by Advanced Automated Peptide ProteinTechnologies (aapptec), 6309 Shepardsville Rd., Louisville Ky. 40228, ph888 692 9111; www.aapptec.com, and references within. These methodscreate a stable amide linkage attaching the hapten to the spacer.Examples of spacers that can be obtained using known methods andattached to amino-bearing haptens utilizing routine optimization ofreaction conditions employing the methods described and cited above areshown, but not limited to those in Table 2. These spacers allowattachment of the hapten to a thiol group on a carrier.

TABLE 2

Spacers may also be constructed in a step-wise fashion by sequentialattachment of appropriate chemical groups to the hapten including thestep of forming the functional linking group that is capable of bindingto the carrier. See illustrative examples under General ReactionSchemes.

Additionally, when the hapten has a nucleophilic group, for example athiol group, an amino group or a hydroxyl group which will become thepoint of attachment of the spacer, the spacer may also be constructed byalkylation of the thiol, amine or hydroxyl group. Any alkyl group thatis appropriately substituted with a moiety capable of undergoing asubstitution reaction, for example, an alkyl halide, or sulfonic acidester such as p-Toluenesulfonate, may be used to attach the spacer. Manyexamples of alkylation reactions are known to one skilled in the art andspecific examples may be found in the general chemical literature andoptimized through routine experimentation. A discussion of alkylationreactions with many references can be found in Chapter 10 of March'sAdvanced Organic Chemistry, Smith, M. B., and March, J., John Wiley &sons, Inc. NY, 2001. Other linkages may also be employed such asreaction of the nucleophilic moiety, for example an amine, on the haptenwith an isocyanate to form a urea or reaction with an isothiocyanate toform a thiourea linkage, see: Li, Z., et. al., Phosphorus, Sulfur andSilicon and the Related Elements, 2003, 178(2):293-297. Spacers may beattached to haptens bearing hydroxyl groups via reaction with isocyanategroups to form carbamate or urethane linkages. The spacer may bedifferentially activated with the isocyanate functional group on one endand a functional linking group capable of reacting with the carrier,see: Annunziato, M. E., Patel, U.S., Ranade, M. and Palumbo, P.S.,Bioconjugate Chem., 1993, 4:212-218.

For haptens bearing a carboxylic acid group, modes of attachment of aspacer portion to the hapten include activation of the carboxylic acidgroup as an acyl halide or active ester, examples of which are shown inTable 3, preparation of which are described previously, followed byreaction with an amino (—NH₂—), hydrazino (—NH—NH₂—), hydrazido(—C(O)—NH—NH₂—) or hydroxyl group (—OH) on the spacer portion to form anamide, hydrazide, diacylhydrazine or ester linkage, or direct couplingof the carboxylic acid group with an amino group on the spacer portionor directly on the carrier with a peptide coupling reagent and/orcarbodiimide dehydrating reagent, described previously, examples ofwhich are shown in Tables 4 and 5. Procedures found in references citedpreviously for formation of activated esters and use of peptide couplingagents may be employed for attachment of carboxylic acid-bearing haptensto spacer building blocks and protein carriers with available aminogroups utilizing routine optimization of reaction conditions.

TABLE 3

TABLE 4

TABLE 5

Other electrophilic groups may be present on the hapten to attach thespacer, for example, a sulfonyl halide

or electrophilic phosphorous group, for example:

See: Malachowski, William P., Coward, James K., Journal of OrganicChemistry, 1994, 59 (25):7616

or:

R_(c) is alkyl, cycloalkyl, aryl, substituted aryl, aralkyl.

See: Aliouane, L., et. al, Tetrahedron Letters, 2011, 52(28):8681.

Haptens that bear aldehyde or ketone groups may be attached to spacersusing methods including but not limited to reaction with a hydrazidegroup H₂N—NH—C(O)— on the spacer to form an acylhydrazone, see: Chamow,S. M., Kogan, T. P., Peers, D. H., Hastings, R. C., Byrn, R. A. andAskenaszi, A., J. Biol. Chem., 1992, 267(22): 15916. Examples ofbifunctional hydrazide spacer groups that allow attachment to a thiolgroup on the carrier are shown in Table 6.

TABLE 6

Haptens may also contain thiol groups which may be reacted with thecarrier provided that the carrier has been modified to provide a groupthat may react with the thiol. Carrier groups may be modified by methodsincluding but not limited to attachment of a group containing amaleimide functional group by reaction of an amino group on the carrierwith N-Succinimidyl maleimidoacetate, (AMAS, CAS #55750-61-3),Succinimidyl iodoacetate (CAS#151199-81-4), or any of the bifunctionalspacer groups shown in Table 1 to introduce a group which may undergo areaction resulting in attachment of the hapten to the carrier.

The functional linking group capable of forming a bond with the carriermay be any group capable of forming a stable linkage and may be reactiveto a number of different groups on the carrier. The functional linkinggroup may preferably react with an amino group, a carboxylic acid groupor a thiol group on the carrier, or derivative thereof. Non-limitingexamples of the functional linking group are a carboxylic acid group,acyl halide, active ester (as defined previously), isocyanate,isothiocyanate, alkyl halide, amino group, thiol group, maleimide group,acrylate group (H₂C═CH—C(O)—) or vinyl sulfone group H₂C═CH—SO₂—) See:Park, J. W., et. al., Bioconjugate Chem., 2012, 23(3): 350. Thefunctional linking group may be present as part of a differentiallyactivated spacer building block that may be reacted stepwise with thehapten and the resulting hapten derivative may then be reacted with thecarrier. Alternatively, the hapten may be derivatized with a spacer thatbears a precursor group that may be transformed into the functionallinking group by a subsequent reaction. When the functional linkinggroup on the spacer is an amine or a carboxylic acid group, the couplingreaction with the carboxylic acid group or amine on the carrier may becarried out directly through the use of peptide coupling reagentsaccording to procedures in the references cited above for thesereagents.

Particular disulfide groups, for example, pyridyldisulfides, may be usedas the functional linking group on the spacer which may undergo exchangewith a thiol group on the carrier to from a mixed disulfide linkage,see: Ghetie, V., et al., Bioconjugate Chem., 1990, 1:24-31. Thesespacers may be attached by reaction of the amine-bearing hapten with anactive ester which is attached to a spacer bearing the pyridyldisulfidegroup, examples of which include but are not limited to those shown inTable 7.

TABLE 7

Most often the carrier is a protein and the ε-amino groups of the lysineresidues may be used for attachment, either directly by reaction with anamine-reactive functional linking group or after derivitization with athiol-containing group, including N-Succinimidyl S-Acetylthioacetate,(SATA, CAS 76931-93-6), or an analogue thereof, followed by cleavage ofthe actetate group with hydroxylamine to expose the thiol group forreaction with the functional linking group on the hapten. Thiol groupsmay also be introduced into the carrier by reduction of disulfide bondswithin protein carriers with mild reducing reagents including but notlimited to 2-mercaptoethylamine, see: Bilah, M., et. al.,Bioelectrochemistry, 2010, 80(1):49, phosphine reagents, see: Kirley, T.L., Analytical Biochemistry, 1989, 180(2):231 or dithioerythritol (DTT,CAS 3483-12-3) Cleland, W., Biochemistry, 1964, 3:480-482.

General Reaction Schemes

Compounds useful for producing antibodies according to the subjectinvention can be synthesized in accordance with the general syntheticmethods described below. Compounds of Formula (I) can be prepared bymethods known to those who are skilled in the art. The followingreaction schemes are only meant to represent examples of the inventionand are in no way meant to be a limit of the invention.

Compounds of Formula I where R² is CH₂NHC(O)(CH₂)_(m)CO₂H may be madeaccording to Scheme 1. Reaction of(1-methyl-4-(2-methyl-10H-benzo[b]thieno[2,3-e][1,4]diazepin-4-yl)piperazin-2-yl)methanamine,prepared as described in Example 1, Step I, proceeds with a cyclicanhydride compound, such as succinic anhydride or glutaric anhydride, ina solvent such as pyridine, at temperatures ranging from roomtemperature to 60° C., for about 48 hours. Those skilled in the art willrecognize that the same chemistry may be used to create compounds ofFormula I where R¹ is CH₂NHC(O)(CH₂)_(m)CO₂H.

Compounds of Formula I where R² is

may be made according to Scheme 2. Compounds of Formula I, where R² isCH₂NHC(O)(CH₂)_(m)CO₂H, prepared as described in Scheme 1, are treatedwith N-t-butoxycarbonylpiperazine, diethyl cyanophosphonate, and a base,such as diisopropylethylamine. The reaction is carried out in a solvent,such as dichloromethane, for about 2 hours at room temperature.Deprotection of the piperazinyl group is accomplished withtrifluoroacetic anhydride as described in Scheme 2, followed by reactionwith an appropriate anhydride, such as succinic anhydride or maleicanhydride, in the presence of a suitable base such asdiisopropylethylamine. Those skilled in the art will recognize that thesame chemistry may be used to create compounds of Formula I where R¹ is

Compounds of Formula I where R¹ is

may be made according to Scheme 3. The maleimide may be introduced byany method known in the art. Maleimide functionalizing groups such as2,5-dioxopyrrolidin-1-yl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetatewhere m is 1, may be used in a solvent such as DMF or CH₂Cl₂, and abase, such as tributylamine or triethylamine. Alternatively, thedeprotected piperazinyl group described in Scheme 2 may be elaboratedwith a maleimide functionality, as described in Scheme 3 to givecompounds of Formula I where R¹ is

Those skilled in the art will recognize that the same chemistry may beused to create compounds of Formula I where R² is

Compounds in which the spacer and linking group are attached to theunsubstituted secondary nitrogen in the diazepine ring of olanzapine maybe obtained by the reactions depicted in schemes 4 to 8. Acylation ofthe nitrogen is described by Su, J. et. al, Bioorganic and Med. Chem.Letters, 2006, 16:4548. Use of the mono ester mono acid chloride ofsuccinic acid in the presence of a base, under anhydrous conditions inan aprotic solvent, provides an intermediate, the ester functionality ofwhich may be hydrolyzed using standard conditions known to one skilledin the art, for example, aqueous base, to provide a hapten that may befurther elaborated into an immunogen by methods previously describedherein and illustrated by examples of this disclosure.

Su, et. al., above, also report preparation of sulfonamides. Through useof a functionalized sulfonylchloride in the presence of a base, underanhydrous conditions in an aprotic solvent, as shown in Scheme 5, acarboxy hapten may be prepared and transformed into an immunogen bymethods previously described herein and illustrated by examples of thisdisclosure.

Su, et. al., above, also teach methods for preparation of a hydrazine asshown in Scheme 6, through diazotization of the ring nitrogen with anitrite ester followed by reduction with zinc in acetic acid. Theresulting hydrazine may be further functionalized in a number of ways asshown in Scheme 7. Reaction with a bifunctional spacer building block,for example AMAS, in the presence of an amine base, for example,tribuytlamine, in a solvent such as DMF as described elsewhere herein,may provide a maleimide hapten that may be attached to a carrier throughreaction with a thiol group. Sulfonylation in the presence of base witha functionalized sulfonyl chloride, for example,m-carboxybenzenesulfonylchloride may provide a sulfonylhydrazide thatbears a carboxy group for attachment to a carrier by methods previouslydescribed herein and illustrated by examples of this disclosure.Additionally, the hydrazine may be reacted with a functionalizedaldehyde or ketone, for example, levulinic acid, as described in U.S.Pat. No. 4,022,780, with a catalytic amount of acid under conditionswhere water generated by the condensation is removed, to provide ahydrazone as shown in scheme 7. The hydrazone may be subsequentlyreduced using sodium cyanoborohydride in the method of Su, J. et al.,previously referenced, to provide a saturated derivative.

Direct alkylation of the ring nitrogen as shown in Scheme 8, may also beaccomplished using the method described in U.S. Pat. No. 6,034,078 toappend an alkyl group directly to olanzapine. Though use of afunctionalized alkyl halide, for example, 4-chloromethylbutyrate, onemay obtain an intermediate which, through hydrolysis using standardconditions known to one skilled in the art, may provide a hapten thatmay be further elaborated into an immunogen by methods previouslydescribed herein and illustrated by examples of this disclosure.

Maleimide functionalized haptens wherein R¹ or R² is

may be conjugated to proteins according to the method shown in Scheme 9.Activation of protein lysine residues by acylation of theepsilon-nitrogen with N-succinimidyl S-acetylthioacetate (SATA),followed by subsequent hydrolysis of the S-acetyl group withhydroxylamine produces a nucleophilic sulfhydryl group. Conjugation ofthe sulfhydryl activated protein with the maleimide derivatized hapten(prepared as described in general scheme 3) proceeds via a Michaeladdition reaction. Suitable proteins are known to those skilled in theart and include keyhole limpet hemocyanin, bovine thyroglobulin, andovalbumin. The same methodology may be used to conjugate proteins tomaleimide functionalized haptens where R¹ or R² is

Carboxylic acid functionalized haptens, wherein R¹ or R² isCH₂NHC(O)(CH₂)_(m)CO₂H, may be conjugated to proteins according to themethod shown in Scheme 10. Reaction with N-hydroxysuccinimide and asuitable coupling agent, such as dicyclohexylcarbodiimide, and a base,such as tributyl amine, in a solvent such as DMF, at a temperature ofabout 20° C., for about 18 hrs activates the carboxylic acid with thehydroxypyrrolidine-2,5-dione leaving group. The activated linker andhapten may then be conjugated to a protein in a solvent, such as a pH7.5 phosphate buffer, at about 20° C., for about 2.5 hours. Suitableproteins are known to those skilled in the art and include keyholelimpet hemocyanin, bovine thyroglobulin, and ovalbumin. The samemethodology may be used to conjugate proteins to carboxylic acidfunctionalized haptens where R¹ or R² is

Antibodies

The present invention is directed to an isolated antibody or a bindingfragment thereof, which binds to olanzapine and which: (i) is generatedin response to a conjugate of a compound of Formula I and an immunogeniccarrier; or (ii) competes for an epitope which is the same as an epitopebound by the antibody of (i). The term “antibody” refers to a specificprotein capable of binding an antigen or portion thereof (in accordancewith this invention, capable of binding to an anti-psychotic drug ormetabolite thereof). An antibody is produced in response to an immunogenwhich may have been introduced into a host, e.g., an animal or a human,by injection. The generic term “antibody” includes polyclonalantibodies, monoclonal antibodies, and antibody fragments.

“Antibody” or “antigen-binding antibody fragment” refers to an intactantibody, or a fragment thereof, that competes with the intact antibodyfor binding. Generally speaking, an antibody or antigen-binding antibodyfragment, is said to specifically bind an antigen when the dissociationconstant is less than or equal to 1 μM, preferably less than or equal to100 nM and most preferably less than or equal to 10 nM. Binding can bemeasured by methods know to those skilled in the art, an example beingthe use of a BIAcore™ instrument.

Antibody fragments comprise a portion of an intact antibody, preferablythe antigen binding or variable region of the intact antibody. Bindingfragments include Fab, Fab′, F(ab′)₂, and Fv fragments; diabodies;linear antibodies; single-chain antibody molecules; and multispecificantibodies formed from antibody fragments. An antibody other than a“bispecific” or “bifunctional” antibody is understood to have each ofits binding sites identical.

As used herein, “epitope” includes any protein determinant capable ofspecific binding to an immunoglobulin or T-cell receptor. Epitopicdeterminants usually consist of chemically active surface groupings ofmolecules such as amino acids or sugar side chains and usually havespecific three dimensional structural characteristics, as well asspecific charge characteristics. Two antibodies are said to “bind thesame epitope” if one antibody is shown to compete with the secondantibody in a competitive binding assay, by any of the methods wellknown to those skilled in the art (such as the BIAcore™ method referredto above). In reference to a hapten (such as olanzapine or otheranti-psychotic drug), an antibody can be generated against thenon-antigenic hapten molecule by conjugating the hapten to animmunogenic carrier. An antibody is then generated which recognizes an“epitope” defined by the hapten.

“Isolated” when used in the context of an antibody means altered “by thehand of man” from any natural state; i.e., that, if it occurs in nature,it has been changed or removed from its original environment, or both.For example, a naturally occurring antibody naturally present in aliving animal in its natural state is not “isolated”, but the sameantibody separated from the coexisting materials of its natural state is“isolated”, as the term is employed herein. Antibodies may occur in acomposition, such as an immunoassay reagent, which are not naturallyoccurring compositions, and therein remain isolated antibodies withinthe meaning of that term as it is employed herein.

“Cross-reactivity” refers to the reaction of an antibody with an antigenthat was not used to induce that antibody.

Preferably, the antibody of the subject invention will bind to the drugand any desired pharmacologically active metabolites. By altering thelocation of the attachment of the immunogenic carrier to the compoundsof the invention, selectivity and cross-reactivity with metabolites canbe engineered into the antibodies. For olanzapine, cross-reactivity withthe related drug clozapine may or may not be desirable, and crossreactivity with olanzapine metabolites such as 10-N-gluronide or4-N-desmethyl olanzapine may or may not be desirable. Antibodies may begenerated that detect multiple ones of these drugs and/or metabolites,or antibodies may be generated that detect each separately (thusdefining the antibody “specific binding” properties). An antibodyspecifically binds one or more compounds when its binding of the one ormore compounds is equimolar or substantially equimolar.

Methods of producing such antibodies comprise inoculating a host withthe conjugate described herein. Suitable hosts include, but are notlimited to, mice, rats, hamsters, guinea pigs, rabbits, chickens,donkeys, horses, monkeys, chimpanzees, orangutans, gorillas, humans, andany species capable of mounting a mature immune response. Theimmunization procedures are well established in the art and are setforth in numerous treatises and publications including “The ImmunoassayHandbook”, 2nd Edition, edited by David Wild (Nature Publishing Group,2000) and the references cited therein.

Preferably, an immunogen embodying features of the present invention isadministered to a host subject, e.g., an animal or human, in combinationwith an adjuvant. Suitable adjuvants include, but are not limited to,Freund's adjuvant, powdered aluminum hydroxide (alum), aluminumhydroxide together with Bordetella pertussis, and monophosphoryl lipidA-synthetic trehalose dicorynomycolate (MPL-TDM).

Typically, an immunogen or a combination of an immunogen and an adjuvantis injected into a mammalian host by one or multiple subcutaneous orintraperitoneal injections. Preferably, the immunization program iscarried out over at least one week, and more preferably, over two ormore weeks. Polyclonal antibodies produced in this manner can beisolated and purified utilizing methods well know in the art.

Monoclonal antibodies can be produced by the well-established hybridomamethods of Kohler and Milstein, e.g., Nature 256:495-497 (1975).Hybridoma methods typically involve immunizing a host or lymphocytesfrom a host, harvesting the monoclonal antibody secreting or having thepotential to secrete lymphocytes, fusing the lymphocytes to immortalizedcells, and selecting cells that secrete the desired monoclonal antibody.

A host can be immunized to elicit lymphocytes that produce or arecapable of producing antibodies specific for an immunogen.Alternatively, the lymphocytes can be immunized in vitro. If human cellsare desired, peripheral blood lymphocytes can be used, although spleencells or lymphocytes from other mammalian sources are preferred.

The lymphocytes can be fused with an immortalized cell line to formhybridoma cells, a process which can be facilitated by the use of afusing agent, e.g., polyethylene glycol. By way of illustration, mutantrodent, bovine, or human myeloma cells immortalized by transformationcan be used. Substantially pure populations of hybridoma cells, asopposed to unfused immortalized cells, are preferred. Thus, followingfusion, the cells can be grown in a suitable medium that inhibits thegrowh or survival of unfused, immortalized cells, for example, by usingmutant myeloma cells that lack the enzyme hypoxanthine guaninephosphoribosyl transferase (HGPRT). In such an instance, hypoxanthine,aminopterin, and thymidine can be added to the medium (HAT medium) toprevent the growth of HGPRT-deficient cells while permitting hybridomasto grow.

Preferably, immortalized cells fuse efficiently, can be isolated frommixed populations by selection in a medium such as HAT, and supportstable and high-level expression of antibody following fusion. Preferredimmortalized cell lines include myeloma cell lines available from theAmerican Type Culture Collection, Manassas, Va.

Because hybridoma cells typically secrete antibody extracellularly, theculture media can be assayed for the presence of monoclonal antibodiesspecific for the anti-psychotic drug. Immunoprecipitation of in vitrobinding assays, for example, radiioimmunoassay (RIA) or enzyme-linkedimmunosorbent assay (ELISA), can be used to measure the bindingspecificity of monoclonal antibodies.

Monoclonal antibody-secreting hybridoma cells can be isolated as singleclones by limiting dilution procedures and sub-cultured. Suitableculture media include, but are not limited to, Dulbecco's ModifiedEagle's Medium, RPMI-1640, and polypeptide-free, polypeptide-reduced, orserum-free media, e.g., Ultra DOMA PF or HL-1, available fromBiowhittaker, Walkersville, Md. Alternatively, the hybridoma cells canbe grown in vivo as ascites.

Monoclonal antibodies can be isolated and/or purified from a culturemedium or ascites fluid by conventional immunoglobulin (Ig) purificationprocedures including, but not limited to, polypeptide A-SEPHAROSE,hydroxylapatite chromatography, gel electrophoresis, dialysis, ammoniumsulfate precipitation, and affinity chromatography.

Monoclonal antibodies can also be produced by recombinant methods suchas are described in U.S. Pat. No. 4,166,452. DNA encoding monoclonalantibodies can be isolated and sequenced using conventional procedures,e.g., using oligonucleotide probes that specifically bind to murineheavy and light antibody chain genes, preferably to probe DNA isolatedfrom monoclonal antibody hybridoma cells lines secreting antibodiesspecific for anti-psychotic drugs.

Antibody fragments which contain specific binding sites for theanti-psychotic drug may also be generated. Such fragments include, butare not limited to, the F(ab′)₂ fragments which can be produced bypepsin digestion of the antibody molecule and the Fab fragments whichcan be generated by reducing the disulfide bridges of the F(ab′)₂fragments. Alternatively, Fab expression libraries may be constructed toallow rapid and easy identification of monoclonal Fab fragments with thedesired specificity (Huse et al., Science 256:1270-1281 (1989)). Fab, Fvand ScFv antibody fragments can all be expressed in and secreted fromEscherichia coli, allowing for the production of large amounts of thesefragments. Alternatively, Fab′-SH fragments can be directly recoveredfrom E. coli and chemically coupled to form F(ab′)₂ fragments (Carter etal., BioTechnology 10:163-167 (1992)). Other techniques for theproduction of antibody fragments are known to those skilled in the art.Single chain Fv fragments (scFv) are also envisioned (see U.S. Pat. Nos.5,761,894 and 5,587,458). Fv and sFv fragments are the only species withintact combining sites that are devoid of constant regions; thus, theyare likely to show reduced non-specific binding. The antibody fragmentmay also be a “linear antibody” e.g., as described in U.S. Pat. No.5,642,870, for example. Such linear antibody fragments may bemonospecific or bispecific.

Assay Kits and Devices

An assay kit (also referred to as a reagent kit) can also be providedcomprising an antibody as described above. A representative reagent kitmay comprise an antibody that binds to the anti-psychotic drug,olanzapine, a complex comprising an analog of an anti-psychotic drug ora derivative thereof coupled to a labeling moiety, and may optionallyalso comprise one or more calibrators comprising a known amount of ananti-psychotic drug or a related standard.

The phrase “assay kit” refers to an assembly of materials and reagentsthat is used in performing an assay. The reagents can be provided inpackaged combination in the same or in separate containers, depending ontheir cross-reactivities and stabilities, and in liquid or inlyophilized form. The amounts and proportions of reagents provided inthe kit can be selected so as to provide optimum results for aparticular application. An assay kit embodying features of the presentinvention comprises antibodies which bind olanzapine. The kit mayfurther comprise competitive binding partners of olanzapine andcalibration and control materials.

The phrase “calibration and control material” refers to any standard orreference material containing a known amount of an analyte. A samplesuspected of containing an analyte and the corresponding calibrationmaterial are assayed under similar conditions. The concentration ofanalyte is calculated by comparing the results obtained for the unknownspecimen with the results obtained for the standard. This is commonlydone by constructing a calibration curve.

Antibodies embodying features of the present invention can be includedin a kit, container, pack, or dispenser together with instructions fortheir utilization. When the antibodies are supplied in a kit, thedifferent components of the immunoassay may be packaged in separatecontainers and admixed prior to use. Such packaging of the componentsseparately may permit long-term storage without substantiallydiminishing the functioning of the active components. Furthermore,reagents can be packaged under inert environments, e.g., under apositive pressure of nitrogen gas, argon gas, or the like, which isespecially preferred for reagents that are sensitive to air and/ormoisture.

Reagents included in kits embodying features of the present inventioncan be supplied in all manner of containers such that the activities ofthe different components are substantially preserved while thecomponents themselves are not substantially adsorbed or altered by thematerials of the container. Suitable containers include, but are notlimited to, ampules, bottles, test tubes, vials, flasks, syringes,envelopes, e.g., foil-lined, and the like. The containers may becomprised of any suitable material including, but not limited to, glass,organic polymers, e.g., polycarbonate, polystyrene, polyethylene, etc.,ceramic, metal, e.g., aluminum, metal alloys, e.g., steel, cork, and thelike. In addition, the containers may comprise one or more sterileaccess ports, e.g., for access via a needle, such as may be provided bya septum. Preferred materials for septa include rubber andpolytetrafluoroethylene of the type sold under the trade name TEFLON byDuPont (Wilmington, Del.). In addition, the containers may comprise twoor more compartments separated by partitions or membranes that can beremoved to allow mixing of the components.

Reagent kits embodying features of the present invention may also besupplied with instructional materials. Instructions may be printed,e.g., on paper and/or supplied in an electronically-readable medium.Alternatively, instructions may be provided by directing a user to aninternet website, e.g., specified by the manufacturer or distributor ofthe kit and/or via electronic mail.

The antibody may also be provided as part of an assay device. Such assaydevices include lateral flow assay devices. A common type of disposablelateral flow assay device includes a zone or area for receiving theliquid sample, a conjugate zone, and a reaction zone. These assaydevices are commonly known as lateral flow test strips. They employ aporous material, e.g., nitrocellulose, defining a path for fluid flowcapable of supporting capillary flow. Examples include those shown inU.S. Pat. Nos. 5,559,041, 5,714,389, 5,120,643, and 6,228,660 all ofwhich are incorporated herein by reference in their entireties.

Another type of assay device is a non-porous assay device havingprojections to induce capillary flow. Examples of such assay devicesinclude the open lateral flow device as disclosed in PCT InternationalPublication Nos. WO 2003/103835, WO 2005/089082, WO 2005/118139, and WO2006/137785, all of which are incorporated herein by reference in theirentireties.

In a non-porous assay device, the assay device generally has at leastone sample addition zone, at least one conjugate zone, at least onereaction zone, and at least one wicking zone. The zones form a flow pathby which sample flows from the sample addition zone to the wicking zone.Also included are capture elements, such as antibodies, in the reactionzone, capable of binding to the analyte, optionally deposited on thedevice (such as by coating); and a labeled conjugate material alsocapable of participating in reactions that will enable determination ofthe concentration of the analyte, deposited on the device in theconjugate zone, wherein the labeled conjugate material carries a labelfor detection in the reaction zone. The conjugate material is dissolvedas the sample flows through the conjugate zone forming a conjugate plumeof dissolved labeled conjugate material and sample that flows downstreamto the reaction zone. As the conjugate plume flows into the reactionzone, the conjugated material will be captured by the capture elementssuch as via a complex of conjugated material and analyte (as in a“sandwich” assay) or directly (as in a “competitive” assay). Unbounddissolved conjugate material will be swept past the reaction zone intothe at least one wicking zone. Such devices can include projections ormicropillars in the flow path.

An instrument such as that disclosed in US Patent Publication Nos.US20060289787A1 and US 20070231883A1, and U.S. Pat. Nos. 7,416,700 and6,139,800, all of which are incorporated herein by reference in theirentireties, is able to detect the bound conjugated material in thereaction zone. Common labels include fluorescent dyes that can bedetected by instruments which excite the fluorescent dyes andincorporate a detector capable of detecting the fluorescent dyes.

Immunoassays

The antibodies thus produced can be used in immunoassays torecognize/bind to the anti-psychotic drug, thereby detecting thepresence and/or amount of the drug in a patient sample. Preferably, theassay format is a competitive immunoassay format. Such an assay formatand other assays are described, among other places, in Hampton et al.(Serological Methods, A Laboratory Manual, APS Press, St. Paul, Minn.1990) and Maddox et al. (J. Exp. Med. 158:12111, 1983).

The term “analyte” refers to any substance or group of substances, thepresence or amount of which is to be determined. Representativeanti-psychotic drug analytes include, but are not limited to,risperidone, paliperidone, olanzapine, aripiprazole, and quetiapine.

The term “competitive binding partner” refers to a substance or group ofsubstances, such as may be employed in a competitive immunoassay, whichbehave similarly to an analyte with respect to binding affinity to anantibody. Representative competitive binding partners include, but arenot limited to, anti-psychotic drug derivatives and the like.

The term “detecting” when used with an analyte refers to anyquantitative, semi-quantitative, or qualitative method as well as to allother methods for determining an analyte in general, and ananti-psychotic drug in particular. For example, a method that merelydetects the presence or absence of an anti-psychotic drug in a samplelies within the scope of the present invention, as do methods thatprovide data as to the amount or concentration of the anti-psychoticdrug in the sample. The terms “detecting”, “determining”, “identifying”,and the like are used synonymously herein, and all lie within the scopeof the present invention.

A preferred embodiment of the subject invention is a competitiveimmunoassay wherein antibodies which bind the anti-psychotic drug, orthe drug or competitive binding partner thereof, are attached to a solidsupport (such as the reaction zone in a lateral flow assay device) andlabeled drug or competitive binding partner thereof, or labeledantibody, respectively, and a sample derived from the host are passedover the solid support and the amount of label detected attached to thesolid support can be correlated to a quantity of drug in the sample.

Any sample that is suspected of containing an analyte, e.g., ananti-psychotic drug, can be analyzed in accordance with the methods ofthe presently preferred embodiments. The sample can be pretreated ifdesired and can be prepared in any convenient medium that does notinterfere with the assay. Preferably, the sample comprises an aqueousmedium such as a body fluid from a host, most preferably plasma orserum.

It is to be understood that all manner of immunoassays employingantibodies are contemplated for use in accordance with the presentlypreferred embodiments, including assays in which antibodies are bound tosolid phases and assays in which antibodies are in liquid media. Methodsof immunoassays that can be used to detect analytes using antibodiesembodying features of the present invention include, but are not limitedto, competitive (reagent limited) assays wherein labeled analyte(analyte analog) and analyte in a sample compete for antibodies andsingle-site immunometric assays wherein the antibody is labeled; and thelike.

The present invention is further described by the following examples.The examples are provided solely to illustrate the invention byreference to specific embodiments. These exemplifications, whileillustrating certain specific aspects of the invention, do not portraythe limitations or circumscribe the scope of the disclosed invention.

All examples were carried out using standard techniques, which are wellknown and routine to those of skill in the art, except where otherwisedescribed in detail. Routine molecular biology techniques of thefollowing examples can be carried out as described in standardlaboratory manuals, such as Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd Ed., Cold Spring Habor Laboratory Press, ColdSpring Harbor, N.Y. (1989).

Copending applications entitled “Haptens of Aripiprazole” (AttorneyDocket No. PRD3265USPSP, U.S. Provisional Patent Appl. No. 61/691,450,filed Aug. 21, 2012), “Haptens of Olanzapine” (Attorney Docket No.PRD3266USPSP, U.S. Provisional Patent Appl. No. 61/691,454, filed Aug.21, 2012), “Haptens of Paliperidone” (Attorney Docket No. PRD3267USPSP,U.S. Provisional Patent Appl. No. 61/691,459, filed Aug. 21, 2012),“Haptens of Quetiapine” (Attorney Docket No. PRD3268USPSP, U.S.Provisional Patent Appl. No. 61/691,462, filed Aug. 21, 2012), “Haptensof Risperidone and Paliperidone” (Attorney Docket No. PRD3269USPSP, U.S.Provisional Patent Appl. No. 61/691,469, filed Aug. 21, 2012),“Antibodies to Aripiprazole Haptens and Use Thereof” (Attorney DocketNo. CDS5128USPSP, U.S. Provisional Patent Appl. No. 61/691,544, filedAug. 21, 2012), “Antibodies to Paliperidone Haptens and Use Thereof”(Attorney Docket No. CDS5126USPSP, U.S. Provisional Patent Appl. No.61/691,634, filed Aug. 21, 2012), “Antibodies to Quetiapine Haptens andUse Thereof” (Attorney Docket No. CDS5134USPSP, U.S. Provisional PatentAppl. No. 61/691,598, filed Aug. 21, 2012), “Antibodies to RisperidoneHaptens and Use Thereof” (Attorney Docket No. CDS5130USPSP, U.S.Provisional Patent Appl. No. 61/691,615, filed Aug. 21, 2012),“Antibodies to Aripiprazole and Use Thereof” (Attorney Docket No.CDS5129USPSP, U.S. Provisional Patent Appl. No. 61/691,522, filed Aug.21, 2012), “Antibodies to Olanzapine and Use Thereof” (Attorney DocketNo. CDS5133USPSP, U.S. Provisional Patent Appl. No. 61/691,645, filedAug. 21, 2012), “Antibodies to Paliperidone and Use Thereof” (AttorneyDocket No. CDS5127USPSP, U.S. Provisional Patent Appl. No. 61/691,692,filed Aug. 21, 2012), “Antibodies to Quetiapine and Use Thereof”(Attorney Docket No. CDS5135USPSP, U.S. Provisional Patent Appl. No.61/691,659, filed Aug. 21, 2012), “Antibodies to Risperidone and UseThereof” (Attorney Docket No. CDS5131USPSP, U.S. Provisional PatentAppl. No. 61/691,675, filed Aug. 21, 2012), and “Antibodies toRisperidone and Use Thereof” (Attorney Docket No. CDS5145USPSP, U.S.Provisional Patent Appl. No. 61/790,880, filed Mar. 15, 2013) are allincorporated herein by reference in their entireties.

Example 1(1-Methyl-4-(2-methyl-10H-benzo[b]thieno[2,3-e][1,4]diazepin-4-yl)piperazin-2-yl)methanamine

Step A tert-Butyl 3-cyanopiperazine-1-carboxylate

To a solution of tert-butyl 3-cyanopiperazine-1-carboxylate (21.1 g, 0.1mol) and aqueous formaldehyde (24 g, 37% in water) in THF was addedsodium cyanoborohydride (31.5 g, 0.5 mol) in small portions. Thereaction mixture was aged at ambient temperature overnight then dilutedwith water and extracted with ethyl acetate. The organic phase waswashed with saturated aqueous sodium chloride, dried over anhydroussodium sulfate, filtered, and concentrated under vacuum. The crudeproduct was purified by column chromatography to provide the titlecompound. ¹H NMR (400 MHz, MeOD) δ 4.23-4.18 (m, 1H), 4.01-3.97 (br,1H), 3.92-3.90 (br, 1H), 2.92-2.89 (br, 1H), 2.88-2.87 (br, 1H),2.65-2.62 (m, 1H), 2.378 (s, 3H), 2.36-2.33 (m, 1H), 1.47 (s, 9H).

Step B tert-Butyl 3-(aminomethyl)-4-methylpiperazine-1-carboxylate

To a solution of tert-butyl 3-cyano-4-methylpiperazine-1-carboxylate,prepared as described in Step A, (10.5 g, 47 mmol) in methanol (200 mL)was added metallic nickel (10 g) and triethylamine (5 mL). The mixturewas stirred at ambient temperature overnight under atmosphere ofhydrogen gas (50 psi). Upon consumption of tert-butyl3-cyano-4-methylpiperazine-1-carboxylate, the mixture was filtered, andthe filtrate was concentrated under vacuum to provide crude tert-butyl3-(aminomethyl)-4-methylpiperazine-1-carboxylate used in the next stepwithout purification.

Step C tert-Butyl3-((1,3-dioxoisoindolin-2-yl)methyl)-4-methylpiperazine-1-carboxylate

To a mixture of tert-butyl3-(aminomethyl)-4-methylpiperazine-1-carboxylate, prepared as describedin the previous step, (5.5 g, crude) and sodium bicarbonate (2.52 g, 30mmol) in tetrahydrofuran (100 mL) was added a solution of2H-isoindole-2-carboxylic acid, 1,3-dihydro-1,3-dioxo-, ethyl ester(6.59 g, 30 mmol) in tetrahydrofuran (20 mL) at ambient temperature.After stirring for 30 minutes, the suspension was filtered, and thefiltrate was concentrated to give crude product which was purified bycolumn chromatography to provide the title compound. ¹H NMR (400 MHz,MeOD) δ 7.87-7.85 (m, 2H), 7.87-7.80 (m, 2H), 3.94-3.90 (m, 1H),3.75-3.65 (br, 3H), 3.43-3.41 (br, 1H), 3.30-3.28 (m, 2H), 3.49 (s, 3H),2.39-2.38 (m, 1H), 2.30-2.28 (m, 1H), 1.36 (s, 9H).

Step D 2-((1-Methylpiperazin-2-yl)methyl)isoindoline-1,3-dione

A solution of tert-butyl3-((1,3-dioxoisoindolin-2-yl)methyl)-4-methylpiperazine-1-carboxylate,prepared as described in the previous step, (8.6 g) in methanolichydrogen chloride (20 mL) was stirred at room temperature for 1 hour.The solvent was removed under vacuum to provide2-((1-methylpiperazin-2-yl)methyl)isoindoline-1,3-dione which was usedin the next step without further purification. ¹H NMR (400 MHz, MeOD) δ7.88-7.86 (m, 2H), 7.82-7.80 (m, 2H), 3.99-3.95 (m, 1H), 3.77-3.73 (m,1H), 3.24-3.23 (m, 1H), 3.29-3.23 (m, 1H), 3.17-3.14 (m, 1H), 3.04-2.84(m, 2H), 2.81-2.78 (m, 1H), 2.55 (s, 3H), 2.46-2.40 (m, 1H).

Step E 5-Methyl-2-((2-nitrophenyl)amino)thiophene-3-carbonitrile

To a solution of 2-amino-5-methylthiophene-3-carbonitrile (13.8 g, 100mmol) and 1-fluoro-2-nitrobenzene (16.92 g, 120 mmol) indimethylsulfoxide was added potassium hydroxide (11.2 g, 200 mmol). Thereaction mixture was stirred at room temperature overnight. The mixturewas diluted with water, and the resulting suspension was filtered. Thefiltered cake was dried to give5-methyl-2-((2-nitrophenyl)amino)thiophene-3-carbonitrile as a red solidused without further purification. ¹H NMR: (400 MHz, CDCl₃) δ 9.69 (s,1H), 8.27-8.25 (m, 1H), 7.56-7.52 (m, 1H), 7.23-7.20 (m, 1H), 7.0-6.96(m, 1H), 6.80 (s, 1H), 2.49 (s, 3H).

Step F 2-((2-Aminophenyl)amino)-5-methylthiophene-3-carbonitrile

To a solution of5-methyl-2-((2-nitrophenyl)amino)thiophene-3-carbonitrile, prepared asdescribed in the previous step, (43.3 g, 0.157 mol) in ethyl acetate(500 mL) was added 10% palladium on carbon (8 g). The black mixture wasstirred at room temperature overnight under an atmosphere of hydrogengas. When LCMS showed that most of5-methyl-2-((2-nitrophenyl)amino)thiophene-3-carbonitrile was consumedcompletely, the mixture was filtered and the filtrate was concentratedto provide 2-((2-aminophenyl)amino)-5-methylthiophene-3-carbonitrile. ¹HNMR (400 MHz, CDCl₃) δ 7.29-7.21 (m, 1H), 7.11-7.10 (m, 1H), 6.86-6.79(m, 2H), 6.48-6.47 (m, 1H), 6.42 (brs, 1H), 3.75-3.70 (br, 2H), 2.28 (s,3H).

Step G 2-Methyl-10H-benzo[b]thieno[2,3-e][1,4]diazepin-4-amine

A mixture of 2-((2-aminophenyl)amino)-5-methylthiophene-3-carbonitrile,prepared as described in the previous step, (22.9 g, 100 mmol) inisopropanol (150 mL) and aqueous hydrochloric acid (50 mL, 18%) washeated at 80° C. for 3 hrs. The resulting suspension was filtered andthe filter cake was dried to give the title compound as a red solid. ¹HNMR (400 MHz CDCl₃) δ 7.14-7.12 (t, 1H), 7.7.12-7.10 (t, 1H), 6.95-6.93(d, J=8 MHz, 1H), 6.81-6.79 (d, J=8 MHz, 1H), 6.70 (s, 1H), 2.30 (s,3H).

Step H2-((1-Methyl-4-(2-methyl-10H-benzo[b]thieno[2,3-e][1,4]diazepin-4-yl)piperazin-2-yl)methyl)isoindoline-1,3-dione

A solution of 2-((1-methylpiperazin-2-yl)methyl)isoindoline-1,3-dione,prepared as described in step D, (100 mg, 0.38 mmol),2-methyl-10H-benzo[b]thieno[2,3-e][1,4]diazepin-4-amine, prepared asdescribed in step G, (150 mg, 0.52 mmol) and diisopropylethylamine (0.49g, 3.8 mmol) in dimethylsulfoxide (0.5 mL) was stirred at 170° C. for 2hrs. The reaction was diluted with water and extracted with ethylacetate. The organic phase was concentrated and the residue purified bycolumn to give 15 mg of24(1-methyl-4-(2-methyl-10H-benzo[b]thieno[2,3-e][1,4]diazepin-4-yl)piperazin-2-yl)methyl)isoindoline-1,3-dione.¹H NMR (400 MHz, CDCl₃) δ 7.76-7.73 (m, 1H), 7.45-7.35 (m, 3H),7.18-7.17 (m, 1H), 6.98-6.95 (m, 2H), 6.75-6.73 (m, 1H), 6.46 (s, 1H),4.28-4.25 (m, 1H), 3.96-6.92 (m, 1H), 3.71-3.64 (m, 3H), 3.47-3.41 (m,1H), 3.29-3.28 (m, 1H), 3.12-3.09 (m, 1H), 2.87-2.86 (m, 1H), 2.67-2.53(m, 3H), 2.28 (s, 3H).

Step I(1-Methyl-4-(2-methyl-10H-benzo[b]thieno[2,3-e][1,4]diazepin-4-yl)piperazin-2-yl)methanamine

A solution of2-((1-methyl-4-(2-methyl-10H-benzo[b]thieno[2,3-e][1,4]diazepin-4-yl)piperazin-2-yl)methyl)isoindoline-1,3-dione,prepared as described in the previous step, (1.0 g) in ethanolicmethylamine (20 mL) was stirred at ambient temperature overnight. Thesolvent was removed under vacuum and the residue purified by HPLC togive the hydrochloride salt of(1-methyl-4-(2-methyl-10H-benzo[b]thieno[2,3-e][1,4]diazepin-4-yl)piperazin-2-yl)methanamineas a red solid. ¹H NMR (400 MHz, MeOD) δ 7.46-7.44 (m, 1H), 7.31-7.48(m, 1H), 7.19-7.15 (m, 1H), 6.97-6.95 (m, 1H), 6.74 (s, 1H), 4.80-4.71(br, 1H), 4.28-4.20 (br, 2H), 4.07-4.04 (br, 2H), 3.82-3.70 (br, 3H),3.53-3.48 (m, 1H), 3.18 (s, 3H), 2.42 (m, 3H); ESI-MS (M+1): 342 calc.for C18H23N5S Exact Mass: 341.17.

Example 22-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((1-methyl-4-(2-methyl-10H-benzo[b]thieno[2,3-e][1,4]diazepin-4-yl)piperazin-2-yl)methyl)acetamide

To a solution of(1-methyl-4-(2-methyl-10H-benzo[b]thieno[2,3-e][1,4]diazepin-4-yl)piperazin-2-yl)methanamine,prepared as described in Example 1, (10.3 mg, 30.2 μmoles) in 570 μL ofDMF and 13.3 μL of tributylamine was added 760 μL of a DMF solution ofN-(α-maleimidoacetoxy)succinimide ester (AMAS, 10 mg/mL, 7.6 mg, 30.2μmoles). The resulting solution was allowed to stir for 18 hours at 20°C., then used as such in conjugation reactions with thiol-activatedprotein.

Example 3(2-Methyl-4-(4-methylpiperazin-1-yl)-10H-benzo[b]thieno[2,3-e][1,4]diazepin-7-yl)methanamine

Step A 2-(4-Cyano-2-nitro-phenylamino)-5-methyl-thiophene-3-carbonitrile

To a suspension of sodium hydride (60%, 0.58 g) in THF (2 mL), was added4-fluoro-3-nitro-benzonitrile (1.33 g, 8.0 mmol) and2-amino-5-methyl-thiophene-3-carbonitrile (1.10 g, 8.0 mmol) in THF (10mL), dropwise. The mixture was stirred at room temperature overnight.Two more batches of sodium hydride (60%, 0.50 g and 0.4 g) were addedover the next 6 hours. After stirring for 3 days, the mixture was pouredinto ice-water (20 mL) and acidified to pH 3 with 6N hydrochloric acid(7 mL). The precipitate was filtered and washed with water. The solidwas extracted with dichloromethane (35 mL). The solution wasconcentrated to a solid, and used in the next step without additionalpurification. LC-MS: m/z 285 (M+1), 307 (M+23). ¹H NMR (CDCl₃, 400 MHz):δ (ppm) 9.76 (s, 1H), 8.59 (s, 1H), 7.70 (d, 1H), 7.14 (d, 1H), 6.87 (s,1H), 2.52 (s, 1H).

Step B10-Amino-2-methyl-4H-3-thia-4,9-diaza-benzo[f]azulene-7-carbonitrilehydrochloride

To a suspension of2-(4-Cyano-2-nitro-phenylamino)-5-methyl-thiophene-3-carbonitrile,prepared as described in the previous step, (0.52 g) in ethanol (5 mL),was added tine chloride (1.36 g, 7.2 mmol) in 6 N HCl. The mixture washeated in an 85° C. oil bath for 3 hours and then cooled in ice bath.The solid was filtered, washed with water, and dried to brown give thetitle compound as a brown solid containing inorganic salt, which wasused in the next step without additional purification. LC-MS: m/z 255(M+1 of free base). ¹H NMR (DMSO-d₆, 400 MHz): δ (ppm) 11.18 (br, 1H),10.09 (s, 1H), 9.35 (br, 1H), 8.94 (br, 1H), 7.54 (d, 1H), 7.27 (s, 1H),6.95 (d, 1H), 2.26 (s, 3H).

Step C2-Methyl-10-(4-methyl-piperazin-1-yl)-4H-3-thia-4,9-diaza-benzo[f]azulene-7-carbonitrile

To a solution of10-amino-2-methyl-4H-3-thia-4,9-diaza-benzo[f]azulene-7-carbonitrilehydrochloride, prepared as described in the previous step, (0.6 g) inDMSO (6 mL) and toluene (6 mL), was added 1-methylpiperazine (4 mL). Themixture was heated in a 130° C. oil bath for 17 hours. The solution wasconcentrated, diluted with ethyl acetate (50 mL), washed with water (20mL) and brine (20 mL), and then concentrated. The solid was dissolved indichloromethane (10 mL) and treated with saturated sodium bicarbonatesolution. The title compound was collected as a light yellowprecipitate, washed with water and dichloromethane, dried, and used inthe next step without additional purification. LC-MS: m/z 338 (M+1). ¹HNMR (CD₃OD, 400 MHz): δ (ppm) 7.19-7.15 (m, 2H), 6.74 (d, 1H), 6.37 (s,1H), 3.51 (m, 4H), 2.53 (m, 4H), 2.34 (s, 3H), 2.32 (s, 3H).

Step D(2-Methyl-4-(4-methylpiperazin-1-yl)-10H-benzo[b]thieno[2,3-e][1,4]diazepin-7-yl)methanamine

To a solution of2-Methyl-10-(4-methyl-piperazin-1-yl)-4H-3-thia-4,9-diaza-benzo[f]azulene-7-carbonitrile,prepared as described in the previous step, (0.25 g) in methanol (90 mL)was added concentrated HCl (0.4 mL) and Pd black (57 mg). Hydrogenationwas carried out at 50 psi for 1 h. More Pd black (147 mg) was added. Themixture was shaken at 50 psi for 22 h. The catalyst was filtered andwashed with methanol. The filtrate was concentrated, treated withsaturated sodium bicarbonate solution (5 mL), and concentrated todryness. The product was purified by silica column. LC-MS: m/z 342(M+1). ¹H NMR (CD₃OD, 400 MHz): δ (ppm) 6.89-6.85 (m, 2H), 6.64 (d, 1H),6.34 (d, 1H), 3.66 (s, 2H), 3.46 (m, 4H), 2.54 (m, 4H), 2.34 (s, 3H),2.30 (d, 3H).

Example 42-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((2-methyl-4-(4-methylpiperazin-1-yl)-10H-benzo[b]thieno[2,3-e][1,4]diazepin-7-yl)methyl)acetamide

To a solution of(2-methyl-4-(4-methylpiperazin-1-yl)-10H-benzo[b]thieno[2,3-e][1,4]diazepin-7-yl)methanamine,prepared as described in Example 3, (3.5 mg, 10.2 μmoles) in 185 μL ofDMF and 4.5 μL of tributylamine was added 260 μL of a DMF solution ofN-(α-maleimidoacetoxy)succinimide ester (AMAS, 10 mg/mL, 2.6 mg, 10.2μmoles). The resulting solution was allowed to stir for 90 minutes at20° C., then used as such in conjugation reaction with thiol-activatedprotein.

Example 56-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((2-methyl-4-(4-methylpiperazin-1-yl)-10H-benzo[b]thieno[2,3-e][1,4]diazepin-7-yl)methyl)hexanamide

To a solution of(2-methyl-4-(4-methylpiperazin-1-yl)-10H-benzo[b]thieno[2,3-e][1,4]diazepin-7-yl)methanamine,prepared as described in Example 3, (59 mg, 0.17 mmol) indichloromethane (4 mL) was added triethylamine (0.048 mL, 0.34 mmol) and6-maleimidohexanoic N-hydroxysuccinimide ester (53 mg, 0.17 mmol) indichloromethane (1 mL). The solution was stirred at room temperature for40 min, then loaded onto a silica column, eluted with 3-5%methanol/dichloromethane containing triethylamine. The title compoundwas obtained as a yellow solid. LC-MS: m/z 535 (M+1).

Example 6N-[2-Methyl-10-(4-methyl-piperazin-1-yl)-4H-3-thia-4,9-diaza-benzo[f]azulen-7-ylmethyl]-succinamicacid

Step A Succinic acid 2,5-dioxo-pyrrolidin-1-yl ester methyl ester

To a solution of 1-hydroxy-pyrrolidine-2,5-dione (1.23 mL, 10 mmol) inethyl acetate (50 mL) was added 3-chlorocarbonyl-propionic acid methylester (1.15 g, 10 mmol). The mixture was cooled in an ice bath.Triethylamine (1.4 mL, 10 mmol) was added dropwise. The resultingsuspension was stirred for 10 min in an ice bath and for 5 min withoutice bath. The white solid was removed by filtration and washed withethyl acetate (3×3 mL). The filtrate was concentrated to a white solid(2.32 g).

Step BN-[2-Methyl-10-(4-methyl-piperazin-1-yl)-4H-3-thia-4,9-diaza-benzo[f]azulen-7-ylmethyl]-succinamicacid methyl ester

To a solution of(2-methyl-4-(4-methylpiperazin-1-yl)-10H-benzo[b]thieno[2,3-e][1,4]diazepin-7-yl)methanamine,prepared as described in Example 3, (40 mg, 0.12 mmol) indichloromethane (2 mL) was added triethylamine (0.030 mL, 0.22 mmol) andsuccinic acid 2,5-dioxo-pyrrolidin-1-yl ester methyl ester, prepared asdescribed in the previous step, (31 mg, 0.13 mmol). The solution wasstirred at room temperature for 1 hour and concentrated. The crude wasloaded onto a silica column, eluted with 3-5% methanol/dichloromethanecontaining ammonium hydroxide to give the title compound as a yellowsolid. LC-MS: m/z 456 (M+1).

Step CN-[2-Methyl-10-(4-methyl-piperazin-1-yl)-4H-3-thia-4,9-diaza-benzo[f]azulen-7-ylmethyl]-succinamicacid

To a suspension ofN-[2-methyl-10-(4-methyl-piperazin-1-yl)-4H-3-thia-4,9-diaza-benzo[f]azulen-7-ylmethyl]-succinamicacid methyl ester, prepared as described in the previous step, (80 mg,0.18 mmol) in THF (1.5 mL) was added LiOH (14 mg) in water (0.5 mL). Thesolution was stirred at room temperature for 3 h, acidified with diluteHCl, and concentrated to dryness. LC-MS: m/z 442 (M+1 of the parent).

Example 72-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((1-methyl-4-(2-methyl-10H-benzo[b]thieno[2,3-e][1,4]diazepin-4-yl)piperazin-2-yl)methyl)acetamide-keyholelimpet hemocyanin-conjugate Step A

To a 3.19 mL solution of keyhole limpet hemocyanin (KLH, 15.2 mg, 0.152μmoles) in 100 mM phosphate buffer, 0.46M sodium chloride, at pH 7.4 wasadded 70.3 μL of a DMF solution of N-succinimidyl-5-acetylthioacetate(SATA, 25 mg/mL, 1.75 mg, 7.60 μmoles). The resulting solution wasincubated at 20° C. for 1 hour on a roller mixer. To the reaction wasadded 319 μL of 2.5M hydroxylamine, 50 mM EDTA, pH 7.0 and the resultingsolution was incubated at 20° C. for 25 min, on a roller mixer. Thereaction was purified on a Sephadex G-25 column using 100 mM phosphatebuffer, 0.46 M sodium chloride, 5 mM EDTA, at pH 6.0.

Step B

To the KLH-SH, prepared as described in the previous step, (4.29 mL,12.7 mg 0.127 μmoles) was added an aliquot of the solution prepared inExample 2, (566.6 μL, 12.7 μmoles). The resulting cloudy mixture wasincubated for 2 hours at 20° C. on a roller mixer. The reaction wasfiltered through a 20 μm syringe filter then purified on a Sephadex G-25column using 100 mM phosphate buffer, 0.46M sodium chloride, at pH 7.4.

Example 82-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((1-methyl-4-(2-methyl-10H-benzo[b]thieno[2,3-e][1,4]diazepin-4-yl)piperazin-2-yl)methyl)acetamide-bovinethyroglobulin-conjugate Step A

To 2.0 mL of a solution of bovine thyroglobulin (BTG, 20.0 mg, 0.03μmoles) in 100 mM phosphate buffer pH 7.5 was added 276.0 μL of a DMFsolution of N-succinimidyl-5-acetylthioacetate (SATA, 25 mg/mL, 6.9 mg,30.0 μmoles). The resulting solution was incubated at 20° C. for 1 houron a roller mixer. To the reaction was added 230 μL of 2.5 Mhydroxylamine, 50 mM EDTA, pH 7.0. The resulting solution was incubatedat 20° C. for 15 minutes on a roller mixer. The reaction was purified ona Sephadex G-25 column using 100 mM phosphate buffer, 5 mM EDTA, at pH6.0.

Step B

To the BTG-SH, prepared as described in the previous step, (4.73 mL,14.3 mg, 0.022 μmoles) was added an aliquot of the solution prepared inExample 2, (969.6 μL, 21.7 μmoles). The resulting cloudy mixture wasincubated for 3 hours at 20° C. on a roller mixer. The reaction wasfiltered through a 0.45 μm syringe filter, then purified on a SephadexG-25 column using 100 mM phosphate buffer, 0.14M sodium chloride, at pH7.4.

Example 92-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((1-methyl-4-(2-methyl-10H-benzo[b]thieno[2,3-e][1,4]diazepin-4-yl)piperazin-2-yl)methyl)acetamide-ovalbumin-conjugateStep A

To 1.2 mL of a solution of ovalbumin (12.0 mg, 0.27 μmoles) in 100 mMphosphate buffer pH 7.5 was added 50.1 μL of a DMF solution ofN-succinimidyl-S-acetylthioacetate (SATA, 25 mg/mL, 1.25 mg, 5.42μmoles). The resulting solution was incubated at 20° C. for 1 hour on aroller mixer. To the reaction was added 120 μL of 2.5M hydroxylamine, 50mM EDTA, at pH 7.0. The resulting solution was incubated at 20° C. for15 minutes on a roller mixer. The reaction was purified on a SephadexG-25 column using 100 mM phosphate buffer, 5 mM EDTA, at pH 6.0.

Step B

To the ovalbumin-SH, prepared as described in the previous step, (4.2mL, 8.0 mg, 0.18 μmoles) was added an aliquot of the solution preparedin Example 2, (200 μL, 4.5 μmoles). The resulting mixture was incubatedfor 3 hours at 20° C. on a roller mixer. The reaction was purified on aSephadex G-25 column using 100 mM phosphate buffer, 0.14M sodiumchloride, at pH 7.4.

Example 102-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((2-methyl-4-(4-methylpiperazin-1-yl)-10H-benzo[b]thieno[2,3-e][1,4]diazepin-7-yl)methyl)acetamide—KeyholeLimpet Hemocyanin—Conjugate

To the KLH-SH, prepared as described in Example 7 Step A, (3.31 mL, 9.8mg, 0.098 μmoles) was added a 300 μL aliquot of2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((2-methyl-4-(4-methylpiperazin-1-yl)-10H-benzo[b]thieno[2,3-e][1,4]diazepin-7-yl)methyl)acetamidesolution, prepared as described in Example 4, (6.9 μmoles). Theresulting cloudy mixture was incubated for 2.5 hours at 20° C. on aroller mixer. The reaction was filtered through a 0.2 μm syringe filterthen purified on a Sephadex G-25 column using 100 mM phosphate buffer,0.46 M sodium chloride, at pH 7.4.

Example 112-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((2-methyl-4-(4-methylpiperazin-1-yl)-10H-benzo[b]thieno[2,3-e][1,4]diazepin-7-yl)methyl)acetamide-ovalbumin-conjugate

To the ovalbumin-SH, prepared as described in Example 9 Step A, (5.38mL, 17.8 mg, 0.40 μmoles) was added a 200 μL aliquot of2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((2-methyl-4-(4-methylpiperazin-1-yl)-10H-benzo[b]thieno[2,3-e][1,4]diazepin-7-yl)methyl)acetamidesolution, prepared as described in Example 4, (10.2 μmoles). Theresulting mixture was incubated for 3 hours at 20° C. on a roller mixer.The reaction was filtered through a 0.45 μm syringe filter then purifiedon a Sephadex G-25 column using 100 mM phosphate buffer, 0.14 M sodiumchloride, at pH 7.4.

Example 12N-[2-Methyl-10-(4-methyl-piperazin-1-yl)-4H-3-thia-4,9-diaza-benzo[f]azulen-7-ylmethyl]-succinamicacid-bovine thyroglobulin-conjugate Step A

A solution ofN-[2-Methyl-10-(4-methyl-piperazin-1-yl)-4H-3-thia-4,9-diaza-benzo[f]azulen-7-ylmethyl]-succinamicacid, prepared as described in Example 6, (7.9 mg, 18.0 μmoles),N-hydroxysuccinimide (NHS, 8.3 mg, 72.0 μmoles) andN,N-dicyclohexylcarbodiimide (14.9 mg, 72.0 μmoles) in 500 μL of DMF and5 μL of tributylamine was allowed to stir for 18 hours at 20° C., thenused as such in conjugation with protein.

Step B

To 2.98 mL of a solution of bovine thyroglobulin (BTG, 14.9 mg, 0.023μmoles) in 100 mM phosphate buffer pH 7.5 was added 500 μL of thesolution prepared in Step A (18.0 μmoles). The resulting cloudy mixturewas incubated at 20° C. for 2.5 hours on a roller mixer. The reactionwas filtered through a 0.45 μm syringe filter then purified on aSephadex G-25 column using 100 mM phosphate buffer, 0.14 M sodiumchloride, at pH 7.4.

Example 13 Competitive Immunoassays for Olanzapine and MultiplexCompetitive Immunoassay for Aripiprazole, Olanzapine, Quetiapine, andRisperidone/Paliperidone

Following a series of immunizations with olanzapine immunogens havingFormulas II and III, mouse tail bleeds were tested for reactivity usingan ELISA. Hybridoma supernatants were also tested, and the ELISA datashown in Tables 8 (hybridomas generated against an olanzapine immunogenhaving Formula II) and 9 (hybridomas generated against an olanzapineimmunogen having Formula III) below shows reactivity of severalhybridomas (fusion partner was NSO cells).

TABLE 8 Plate 2 Dilution 1 2 3 4 5 6 7 8 9 10 11 12 1/400 25 26 27 28 2930 31 21 33 34 35 36 Ag = Bt-Compound#11 1/1200 1/3600 1/10800 1/400 3738 39 40 41 42 43 44 45 46 47 48 1/1200 1/3600 1/10800 1/400 0.01360.0432 0.131 0.0654 0.4092 0.039 0.016 0.1408 0.0712 1.4854 2.00860.0861 Ag = Bt-Compound#11 1/1200 0.0113 0.0194 0.0477 0.0291 0.12930.031 0.012 0.0374 0.0126 0.4411 0.8874 0.0362 1/3600 0.0092 0.01180.0233 0.0153 0.0462 0.013 0.009 0.0314 0.0275 0.2073 0.3555 0.02171/10800 0.0105 0.0111 0.0159 0.0107 0.0224 0.012 0.009 0.0172 0.01680.0972 0.147 0.0141 1/400 0.0333 0.1512 1.1412 1.0762 0.3042 0.04 0.4490.1619 1.8038 0.0933 0.7666 1.258 1/1200 0.0144 0.055 0.4575 0.32230.0907 0.016 0.144 0.0402 0.1536 0.0288 0.2956 0.4374 1/3600 0.0080.0333 0.2036 0.1077 0.0361 0.011 0.051 0.0206 0.708 0.0165 0.12120.2072 1/10800 0.0109 0.0181 0.0885 0.0581 0.027 0.01 0.045 0.02170.5338 0.0132 0.0585 0.0954

TABLE 9 Plate 3 Dilution 1 2 3 4 5 6 7 8 9 10 11 12 100 1B2 1G4 2B3 2G53A3 3E9 3F11 4G9 5G11 8G2 *13 Empty 100 300 300 900 900 2700 2700 1000.0628 0.5634 2.9998 1.9083 0.7869 2.7554 2.296 1.027 0.1174 0.82230.041 0 100 0.0527 0.429 2.7862 1.3797 0.6534 2.3072 2.0249 0.934 0.11150.7692 0.0386 0.0057 300 0.0202 0.1452 1.3705 0.5961 0.2337 1.39630.8952 0.2999 0.0378 0.2486 0.0177 0.0031 300 0.0208 0.1408 1.31660.5236 0.2173 1.1112 0.9114 0.3116 0.0406 0.2483 0.0174 0.0052 9000.0132 0.0242 0.4926 0.1967 0.0849 0.4472 0.2986 0.0896 0.0179 0.08510.012 0.0039 900 0.0148 0.0554 0.4551 0.1731 0.0839 0.4471 0.3499 0.09510.018 0.0863 0.0128 0.0055 2700 0.0109 0.0259 0.1877 0.0713 0.03340.1709 0.1381 0.0362 0.0111 0.036 0.0094 0.0041 2700 0.0122 0.028 0.18350.0903 0.0404 0.1924 0.1502 0.0409 0.0113 0.0325 0.0094 0.005

Supernatant was then tested by competition ELISA to determine if thesignals were specific to olanzapine. FIGS. 1-3 show the results fromthree representative hybridomas resulting from mouse fusion 11.1(olanzapine immunogen having Formula II). Data shows specific reactivityto olanzapine with varied reactivity to clozapine.

FIG. 4 shows the competitive immunoassay format used on a lateral flowassay device in which the capture antibody, an olanzapine clone, wasdeposited on a chip along with a detection conjugate consisting ofolanzapine conjugated to a fluorophore. In this competitive format asshow in FIG. 4, a low level of analyte (olanzapine) results in highsignal, whereas a high level of analyte (olanzapine) results in lowsignal. The amount of olanzapine in the sample can be calculated fromthe loss of fluorescence compared to a control sample with no drugpresent. A typical dose response curve generated with olanzapine clone35 is shown in FIG. 5, with olanzapine clone 61 is shown in FIG. 6, andwith olanzapine clone 3F11 is shown in FIG. 7.

FIG. 8 shows the chip design of a lateral flow assay device according toone embodiment of the subject invention. The device includes a zone orarea for receiving the sample, a conjugate zone (which contains desiredlabeled competitive binding partner(s)), and a reaction zone (eightareas within the reaction zone are indicated; each area can contain aseparate desired antibody). Sample flows from the sample zone throughthe conjugate zone and to the reaction zone.

FIGS. 9-12 show typical dose response curves for an aripiprazolepositive control (sample containing aripiprazole) generated withantibody 5C7 deposited in reaction zone 2 and a labeled aripiprazolecompetitive binding partner in the conjugate zone (FIG. 9), anolanzapine positive control (sample containing olanzapine) generatedwith antibody 4G9-1 deposited in reaction zone 4 and a labeledolanzapine competitive binding partner in the conjugate zone (FIG. 10),a quetiapine positive control (sample containing quetiapine) generatedwith antibody 11 deposited in reaction zone 6 and a labeled quetiapinecompetitive binding partner in the conjugate zone (FIG. 11), and arisperidone positive control (sample containing risperidone) generatedwith antibody 5-9 deposited in reaction zone 8 and a labeled risperidonecompetitive binding partner in the conjugate zone (FIG. 12). The labeledcompetitive binding partners in the conjugate zone compete with thedrugs present in the samples for binding to the antibodies. The amountof label is detected and is an indication of the amount of drug presentin the sample (the amount of signal being inversely proportional to theamount of drug in the sample—see FIG. 4).

In order to confirm that conjugates of labeled competitive bindingpartners do not bind to antibodies deposited in the reaction zones,negative controls were conducted by using samples containing no drugs.Referring to Table 10, a sample containing no aripiprazole is depositedin the sample zone and moves by capillary action through the conjugatezone (this time containing labeled olanzapine, labeled quetiapine, andlabeled risperidone, but no labeled aripiprazole) and to the reactionzone. The reaction zone again contains aripiprazole antibody (5C7) inreaction zone 2. Table 10 below shows the results, confirming that thereis no dose response and the olanzapine, quetiapine, and risperidoneconjugates that move by capillary action through the reaction zone donot bind to the aripiprazole antibody.

TABLE 10 Aripiprazole-Clone 5C7-Math Model 1 (0 ng/mL Conc.) ReactionRead Peak Mean Peak Mean Mean Assay-MM Conj Zone Position Area HeightBackground ARIP-MM1 OLAN, QUET, RISP ARIP 2 0.77 1.56 3.99 ARIP-MM1OLAN, QUET, RISP 4 −0.02 0.06 4.14 ARIP-MM1 OLAN, QUET, RISP 6 0.09 0.104.29 ARIP-MM1 OLAN, QUET, RISP 8 0.13 0.12 4.61 Other Conjugates do notbind to Aripiprazole

Referring to Table 11, a sample containing no olanzapine is deposited inthe sample zone and moves by capillary action through the conjugate zone(this time containing labeled aripiprazole, labeled quetiapine, andlabeled risperidone, but no labeled olanzapine) and to the reactionzone. The reaction zone again contains olanzapine antibody (4G9-1) inreaction zone 4. Table 11 below shows the results, confirming that thereis no dose response and the aripiprazole, quetiapine, and risperidoneconjugates that move by capillary action through the reaction zone donot bind to the olanzapine antibody.

TABLE 11 OLAN-Clone 4G9-1-Math Model 1 (0 ng/mL Conc.) Reaction ReadPeak Mean Peak Mean Mean Assay-MM Conj Zone Position Area HeightBackground OLAN-MM1 ARIP, QUET, RISP 2 −0.03 0.05 4.38 OLAN-MM1 ARIP,QUET, RISP OLAN 4 0.74 1.10 4.56 OLAN-MM1 ARIP, QUET, RISP 6 0.06 0.094.79 OLAN-MM1 ARIP, QUET, RISP 8 0.11 0.13 5.17 Other Conjugates do notbind to Olanzapine

Referring to Table 12, a sample containing no quetiapine is deposited inthe sample zone and moves by capillary action through the conjugate zone(this time containing labeled aripiprazole, labeled olanzapine, andlabeled risperidone, but no labeled quetiapine) and to the reactionzone. The reaction zone again contains quetiapine antibody (11) inreaction zone 6. Table 12 below shows the results, confirming that thereis no dose response and the aripiprazole, olanzapine, and risperidoneconjugates that move by capillary action through the reaction zone donot bind to the quetiapine antibody.

TABLE 12 Quetiapine-Clone 11-Math Model 1 (0 ng/mL Conc.) Reaction ReadPeak Mean Peak Mean Mean Assay-MM Conj Zone Position Area HeightBackground QUET-MM1 ARIP, OLAN, RISP 2 −0.01 0.07 3.85 QUET-MM1 ARIP,OLAN, RISP 4 0.01 0.12 4.01 QUET-MM1 ARIP, OLAN, RISP QUET 6 0.03 0.084.24 QUET-MM1 ARIP, OLAN, RISP 8 0.04 0.07 4.56 Other Conjugates do notbind to Quetiapine

Referring to Table 13, a sample containing no risperidone is depositedin the sample zone and moves by capillary action through the conjugatezone (this time containing labeled aripiprazole, labeled olanzapine, andlabeled quetiapine, but no labeled risperidone) and to the reactionzone. The reaction zone again contains risperidone antibody (5-9) inreaction zone 8. Table 13 below shows the results, confirming that thereis no dose response and the aripiprazole, olanzapine, and quetiapineconjugates that move by capillary action through the reaction zone donot bind to the risperidone antibody.

TABLE 13 Risperidone-Clone 5-9-Math Model 1 (0 ng/mL Conc.) ReactionRead Peak Mean Peak Mean Mean Assay-MM Conj Zone Position Area HeightBackground RISP-MM1 ARIP, OLAN, QUET 2 0.02 0.11 7.43 RISP-MM1 ARIP,OLAN, QUET 4 0.05 0.14 7.73 RISP-MM1 ARIP, OLAN, QUET 6 0.20 0.19 8.11RISP-MM1 ARIP, OLAN, QUET RISP 8 1.97 3.23 8.85 Other Conjugates do notbind to Risperidone

In order to confirm that conjugates of labeled competitive bindingpartners bind only to their respective antibodies deposited in thereaction zones, additional negative controls were conducted by againusing samples containing no drugs. Referring to Table 14, a samplecontaining no aripiprazole is deposited in the sample zone and moves bycapillary action through the conjugate zone (this time containinglabeled aripiprazole) and to the reaction zone. The reaction zone againcontains aripiprazole antibody (5C7) in reaction zone 2, as well asolanzapine antibody (4G9-1) in reaction zone 4, quetiapine antibody (11)in reaction zone 6, and risperidone antibody (5-9) in reaction zone 8.Table 14 below shows the results, confirming that there is no doseresponse except to the aripiprazole antibody 5C7 (in reaction zone 2).

TABLE 14 Aripiprazole-Clone 5C7-Math Model 1 (0 ng/mL Conc.) Peak PeakReaction Mean Mean Mean Assay-MM Conj Zone Read Position Area HeightBackground ARIP-MM1 ARIP, OLAN, QUET, RISP ARIP 2 60.34 97.53 5.44ARIP-MM1 ARIP, OLAN, QUET, RISP 4 2.86 3.91 11.66 ARIP-MM1 ARIP, OLAN,QUET, RISP 6 1.12 1.23 11.03 ARIP-MM1 ARIP, OLAN, QUET, RISP 8 3.14 4.1912.94 Only the Aripiprazole Reaction Zone is binding

Referring to Table 15, a sample containing no olanzapine is deposited inthe sample zone and moves by capillary action through the conjugate zone(this time containing labeled olanzapine) and to the reaction zone. Thereaction zone again contains aripiprazole antibody (5C7) in reactionzone 2, as well as olanzapine antibody (4G9-1) in reaction zone 4,quetiapine antibody (11) in reaction zone 6, and risperidone antibody(5-9) in reaction zone 8. Table 15 below shows the results, confirmingthat there is no dose response except to the olanzapine antibody 4G9-1(in reaction zone 4).

TABLE 15 OLAN-Clone 4G9-1-Math Model 1 (0 ng/mL Conc.) Peak PeakReaction Mean Mean Mean Assay-MM Conj Zone Read Position Area HeightBackground OLAN-MM1 ARIP, OLAN, QUET, RISP 2 0.02 0.08 4.86 OLAN-MM1ARIP, OLAN, QUET, RISP OLAN 4 34.23 51.80 5.39 OLAN-MM1 ARIP, OLAN,QUET, RISP 6 0.22 0.32 5.39 OLAN-MM1 ARIP, OLAN, QUET, RISP 8 0.15 0.175.59 Only the Olanzapine Reaction Zone is binding

Referring to Table 16, a sample containing no quetiapine is deposited inthe sample zone and moves by capillary action through the conjugate zone(this time containing labeled quetiapine) and to the reaction zone. Thereaction zone again contains aripiprazole antibody (5C7) in reactionzone 2, as well as olanzapine antibody (4G9-1) in reaction zone 4,quetiapine antibody (11) in reaction zone 6, and risperidone antibody(5-9) in reaction zone 8. Table 16 below shows the results, confirmingthat there is no dose response except to the quetiapine antibody 11 (inreaction zone 6).

TABLE 16 Quetiapine-Clone 11-Math Model 1 (0 ng/mL Conc.) Peak PeakReaction Mean Mean Mean Assay-MM Conj Zone Read Position Area HeightBackground QUET-MM1 ARIP, OLAN, QUET, RISP 2 0.13 0.41 10.02 QUET-MM1ARIP, OLAN, QUET, RISP 4 0.08 0.23 10.47 QUET-MM1 ARIP, OLAN, QUET, RISPQUET 6 140.35 181.33 7.91 QUET-MM1 ARIP, OLAN, QUET, RISP 8 1.58 2.6111.53 Only the Quetiapine Reaction Zone is binding

Referring to Table 17, a sample containing no risperidone is depositedin the sample zone and moves by capillary action through the conjugatezone (this time containing labeled risperidone) and to the reactionzone. The reaction zone again contains aripiprazole antibody (5C7) inreaction zone 2, as well as olanzapine antibody (4G9-1) in reaction zone4, quetiapine antibody (11) in reaction zone 6, and risperidone antibody(5-9) in reaction zone 8. Table 17 below shows the results, confirmingthat there is no dose response except to the risperidone antibody 5-9(in reaction zone 8).

TABLE 17 Risperidone-Clone 5-9-Math Model 1 (0 ng/mL Conc.) Peak PeakReaction Mean Mean Mean Assay-MM Conj Zone Read Position Area HeightBackground RISP-MM1 ARIP, OLAN, QUET, RISP 2 1.03 1.51 9.07 RISP-MM1ARIP, OLAN, QUET, RISP 4 0.65 0.91 9.60 RISP-MM1 ARIP, OLAN, QUET, RISP6 2.61 6.39 10.48 RISP-MM1 ARIP, OLAN, QUET, RISP RISP 8 55.98 100.9111.58 Only the Risperidone Reaction Zone is binding

The results shown above confirm that conjugates of labeled competitivebinding partners bind only to their respective antibodies in thereaction zone.

FIGS. 13-16 show typical dose response curves in specific antibodyreaction zones, and proof of dose response low/high concentration foreach specific assay in the presence of other conjugates. In FIG. 13, asample containing aripiprazole is deposited in the sample zone and movesby capillary action through the conjugate zone (this time containinglabeled aripiprazole, labeled olanzapine, labeled quetiapine, andlabeled risperidone) and to the reaction zone. The reaction zone againcontains aripiprazole antibody (5C7) in reaction zone 2. A typical doseresponse curve was generated as is shown in FIG. 13 only foraripiprazole, and not for olanzapine, quetiapine, or risperidone.

In FIG. 14, a sample containing olanzapine is deposited in the samplezone and moves by capillary action through the conjugate zone (this timecontaining labeled aripiprazole, labeled olanzapine, labeled quetiapine,and labeled risperidone) and to the reaction zone. The reaction zoneagain contains olanzapine antibody (4G9-1) in reaction zone 4. A typicaldose response curve was generated as is shown in FIG. 14 only forolanzapine, and not for aripiprazole, quetiapine, or risperidone.

In FIG. 15, a sample containing quetiapine is deposited in the samplezone and moves by capillary action through the conjugate zone (this timecontaining labeled aripiprazole, labeled olanzapine, labeled quetiapine,and labeled risperidone) and to the reaction zone. The reaction zoneagain contains quetiapine antibody (11) in reaction zone 6. A typicaldose response curve was generated as is shown in FIG. 15 only forquetiapine, and not for aripiprazole, olanzapine, or risperidone.

In FIG. 16, a sample containing risperidone is deposited in the samplezone and moves by capillary action through the conjugate zone (this timecontaining labeled aripiprazole, labeled olanzapine, labeled quetiapine,and labeled risperidone) and to the reaction zone. The reaction zoneagain contains risperidone antibody (5-9) in reaction zone 8. A typicaldose response curve was generated as is shown in FIG. 16 only forrisperidone, and not for aripiprazole, olanzapine, or quetiapine.

FIGS. 17-20 show typical dose response curves for each assay in thepresence of other conjugates and antibodies. In FIG. 17, a samplecontaining aripiprazole is deposited in the sample zone and moves bycapillary action through the conjugate zone (again containing labeledaripiprazole, labeled olanzapine, labeled quetiapine, and labeledrisperidone) and to the reaction zone. The reaction zone again containsaripiprazole antibody (5C7) in reaction zone 2, as well as olanzapineantibody (4G9-1) in reaction zone 4, quetiapine antibody (11) inreaction zone 6, and risperidone antibody (5-9) in reaction zone 8. Atypical dose response curve was generated for aripiprazole, as is shownin FIG. 17. When a sample containing olanzapine was deposited in thesample zone of this chip, a typical dose response curve was generatedfor olanzapine as shown in FIG. 18. When a sample containing quetiapinewas deposited in the sample zone of this chip, a typical dose responsecurve for quetiapine was generated as shown in FIG. 19. When a samplecontaining risperidone was deposited in the sample zone of this chip, atypical dose response curve for risperidone was generated as shown inFIG. 20.

FIGS. 21-24 show comparisons of dose response curves generated aspositive controls (FIGS. 9-12) to dose response curves generated in themultiplex format (FIGS. 17-20). The comparison for aripiprazole is shownin FIG. 21; for olanzapine in FIG. 22; for quetiapine in FIG. 23; andfor risperidone in FIG. 24. These figures show that the positive controlcurves are similar to the multiplex curves.

These data show that a lateral flow assay device of the subjectinvention can be used to detect multiple anti-psychotic drugs using asingle sample from a patient on one portable, point-of-care device.

What is claimed is:
 1. An isolated antibody or a binding fragmentthereof, which binds to olanzapine and which: (i) is generated inresponse to a conjugate of a compound of Formula I and an immunogeniccarrier; or (ii) competes for an epitope which is the same as an epitopebound by an antibody generated in response to a conjugate of a compoundof Formula I and an immunogenic carrier,

wherein: R¹ is H,

CH₂NH₂, CH₂NHC(O)(CH₂)_(m)CO₂H, or Z—(Y)_(p)-G; R² is H,

CH₂NH₂, CH₂NHC(O)(CH₂)_(m)CO₂H, or Z—(Y)_(p)-G; R³ is H, or W—(Y)_(p)-G;provided that two of R¹, R², R³ must be H, and further provided that R¹,R² and R³ may not all be H simultaneously; wherein: Z is selected fromthe group consisting of: —N(R⁴)—, —O—, —S—, -alkyl-, -alkoxyalkyl-,-aminoalkyl-, -thioalkyl-, -heteroalkyl-, alkylcarbonyl-,

wherein: W is selected from the group consisting of: —C(O)—, -alkyl-,-alkoxyalkyl-, -aminoalkyl-, -thioalkyl-, -heteroalkyl-,-alkylcarbonyl-, —N(R⁴)—,

R⁴ is H, an alkyl group, cycloalkyl group, araalkyl group or substitutedor unsubstituted aryl group; Y is an organic spacer group; G is afunctional linking group capable of binding to a carrier; p is 0, or 1;m is 1, 2, 3, 4, or 5; n is 1, 2, 3, 4, or
 5. 2. The antibody of claim1, wherein the antibody is generated in response to a conjugate of acompound of Formula I and an immunogenic carrier.
 3. The antibody ofclaim 1, wherein the antibody fragment is selected from the group offragments consisting of Fv, F(ab′), F(ab′)2, scFv, minibody and diabodyfragments.
 4. The antibody of claim 1, wherein the antibody is amonoclonal antibody.
 5. An assay kit comprising the antibody of claim 1.6. An assay device comprising the antibody of claim
 1. 7. The assaydevice of claim 6 wherein the device is a lateral flow assay device. 8.A method of producing an antibody which binds to olanzapine, the methodcomprising: (i) selecting a host for antibody production; and (ii)inoculating the host with a conjugate of a compound of Formula I and animmunogenic carrier, wherein the host produces an antibody which bindsto olanzapine,

wherein: R¹ is H,

CH₂NH₂, CH₂NHC(O)(CH₂)_(m)CO₂H, or Z—(Y)_(p)-G; R² is H,

CH₂NH₂, CH₂NHC(O)(CH₂)_(m)CO₂H, or Z—(Y)_(p)-G; R³ is H, or W—(Y)_(p)-G;provided that two of R¹, R², R³ must be H, and further provided that R¹,R² and R³ may not all be H simultaneously; wherein: Z is selected fromthe group consisting of: —N(R⁴)—, —O—, —S—, -alkyl-, -alkoxyalkyl-,-aminoalkyl-, -thioalkyl-, -heteroalkyl-, alkylcarbonyl-,

wherein: W is selected from the group consisting of: —C(O)—, -alkyl-,-alkoxyalkyl-, -aminoalkyl-, -thioalkyl-, -heteroalkyl-,-alkylcarbonyl-, —N(R⁴)—,

R⁴ is H, an alkyl group, cycloalkyl group, araalkyl group or substitutedor unsubstituted aryl group; Y is an organic spacer group; G is afunctional linking group capable of binding to a carrier; p is 0, or 1;m is 1, 2, 3, 4, or 5; n is 1, 2, 3, 4, or
 5. 9. A method of producing ahybridoma cell line capable of producing a monoclonal antibody whichbinds to olanzapine, the method comprising: (i) selecting a host forantibody production; (ii) inoculating the host with a conjugate of acompound of Formula I and an immunogenic carrier; (iii) fusing a cellline from said inoculated host with a continuously dividing cell tocreate a fused cell capable of producing a monoclonal antibody whichbinds to olanzapine; and (iv) cloning the fused cell so as to obtain ahybridoma cell line,

wherein: R¹ is H,

CH₂NH₂, CH₂NHC(O)(CH₂)_(m)CO₂H, or Z—(Y)_(p)-G; R² is H,

CH₂NH₂, CH₂NHC(O)(CH₂)_(m)CO₂H, or Z—(Y)_(p)-G; R³ is H, or W—(Y)_(p)-G;provided that two of R¹, R², R³ must be H, and further provided that R¹,R² and R³ may not all be H simultaneously; wherein: Z is selected fromthe group consisting of: —N(R⁴)—, —O—, —S—, -alkyl-, -alkoxyalkyl-,-aminoalkyl-, -thioalkyl-, -heteroalkyl-, alkylcarbonyl-,

wherein: W is selected from the group consisting of: —C(O)—, -alkyl-,-alkoxyalkyl-, -aminoalkyl-, -thioalkyl-, -heteroalkyl-,-alkylcarbonyl-, —N(R⁴)—,

R⁴ is H, an alkyl group, cycloalkyl group, araalkyl group or substitutedor unsubstituted aryl group; Y is an organic spacer group; G is afunctional linking group capable of binding to a carrier; p is 0, or 1;m is 1, 2, 3, 4, or 5; n is 1, 2, 3, 4, or
 5. 10. A method of detectingolanzapine in a sample, the method comprising: (i) contacting a samplewith an antibody of claim 1 labeled with a detectable marker, whereinthe labeled antibody and olanzapine present in the sample form a labeledcomplex; and (ii) detecting the labeled complex so as to detectolanzapine in the sample.
 11. A competitive immunoassay method fordetecting olanzapine in a sample, the method comprising: (i) contactinga sample with the antibody of claim 1, and with olanzapine or acompetitive binding partner of olanzapine, wherein one of the antibodyand the olanzapine or competitive binding partner thereof is labeledwith a detectable marker, and wherein sample olanzapine competes withthe olanzapine or competitive binding partner thereof for binding to theantibody; and (ii) detecting the label so as to detect sampleolanzapine.
 12. The method of claim 11 wherein the olanzapine orcompetitive binding partner thereof is labeled with the detectablemarker.
 13. The method of claim 11 wherein the antibody is labeled witha detectable marker.
 14. The method of claim 11 wherein the immunoassayis performed on a lateral flow assay device and the sample is applied tothe device.
 15. The method of claim 10 or 11, further comprisingdetecting the presence of one or more analytes in addition toolanzapine.
 16. The method of claim 15 wherein the one or more analytesare anti-psychotic drugs other than olanzapine.
 17. The method of claim16 wherein the anti-psychotic drugs other than olanzapine are selectedfrom the group consisting of: risperidone, paliperidone, quetiapine,aripiprazole, and metabolites thereof.
 18. The method of claim 10 or 11,wherein the detection of olanzapine is an indication of patientadherence with prescribed olanzapine therapy.
 19. The method of claim 10or 11, wherein the detection of olanzapine is used to determine whethera patient should be converted from an oral olanzapine regimen to aninjectable anti-psychotic regimen.
 20. The method of claim 10 or 11,wherein the detection of olanzapine is used to determine if the doselevel or dosing interval of oral or injectable olanzapine should beincreased or decreased to ensure attainment or maintenance ofefficacious or safe drug levels.
 21. The method of claim 10 or 11,wherein the detection of olanzapine is an aid in the initiation ofolanzapine therapy by providing evidence of the attainment of minimum pKlevels.
 22. The method of claim 10 or 11, wherein the detection ofolanzapine is used to determine bioequivalence of olanzapine in multipleformulations or from multiple sources.
 23. The method of claim 10 or 11,wherein the detection of olanzapine is used to assess the impact ofpolypharmacy and potential drug-drug interactions.
 24. The method ofclaim 10 or 11, wherein the detection of olanzapine is an indicationthat a patient should be excluded from or included into a clinical trialand is an aid in the subsequent monitoring of adherence to clinicaltrial medication requirements.